Synthesis of a Glycolipidic Amphiphilic Nitrone as a New Spin Trap

Article abstract of DOI:10.1021/jo982343+

The synthesis of a new amphiphilic nitrone, A, derived from a digalactosyl tris(hydroxymethyl)-aminomethane bearing a perfluorocarbon chain is described. A exhibited a surfactant behavior (cmc = 1.6 x 10^-5 mol/L), and the specific recognition o

Full text of DOI:10.1021/jo982343+

3554
J . Org. Chem. 1999, 64, 3554-3556
Syn th esis of a Glycolip id ic Am p h ip h ilic Nitr on e a s a New Sp in
Tr a p
Olivier Ouari,†,‡ Ange Polidori,† Bernard Pucci,*,†,§ Paul Tordo,‡ and Florence Chalier*,‡,|
Laboratoire “Chimie Bioorganique et des Syste`mes Mole´culaires Vectoriels”, Universite´ d’Avignon et des
Pays du Vaucluse, Faculte´ des Sciences, 33, rue Louis Pasteur, 84000-Avignon, France, and
Laboratoire “Structure et Re´activite´ des Espe`ces Paramagne´tiques”, UMR 6517, CNRS et Universite´s
d’Aix-Marseille I et III, Faculte´ de Saint J e´roˆme, case 521, 13397 Marseille Cedex 20, France
Received November 30, 1998
The synthesis of a new amphiphilic nitrone, A, derived from a digalactosyl tris(hydroxymethyl)-
aminomethane bearing a perfluorocarbon chain is described. A exhibited a surfactant behavior
(cmc ) 1.6 × 10-5 mol/L), and the specific recognition of the galactosyl moiety grafted on A by the
KbCWL1 membrane lectin was established. Preliminary experiments showed that A was able to
trap free radicals in aqueous media, the shape of the observed ESR spectra being strongly dependent
upon the nature of the trapped free radical.
Sch em e 1
The spin trapping technique coupled with ESR has
emerged as one of the most useful tools to investigate
the implication of short-lived free radicals in several
clinical disorders.1 Nitrones are the most appropriate spin
traps used in biological systems,2 and among them
R-phenyl-N-tert-butylnitrone (PBN)3 and the 5,5-dimeth-
ylpyrroline N-oxide (DMPO)4 are still the most popular.
In a biological milieu, free radicals can decay by
enzymatic or chemical reactions and their concentrations
always remain very small. This is particularly true in
the case of the superoxide radical in the presence of
superoxide dismutase (SOD) or for the hydroxyl radical
which reacts quickly with most of the biocomponents. To
improve the efficiency of the spin trapping in biological
systems, control of the delivery of the spin trap on the
spot of the radical event is very important. To ensure a
suitable pharmacomodulation, one of the possibilities is
to graft the trapping agent on a natural or a synthetic
carrier. This carrier could modify the hydro- or lipophi-
licity of the active principle and thus would modulate its
membrane crossing ability. Furthermore, it could ensure
specific cell targeting via its functionalization with
ligands such as antibodies, peptides, or carbohydrates.5
As part of our program dealing with the vectorization
and the cell targeting of spin traps,6 we designed a new
glycosidic amphiphilic nitrone, A, derived from PBN
(Scheme 1). The nitronyl group grafted to the tris amino
group by a peptidic spacer arm will ensure the spin
trapping function, and the hydrophilic galactosyl groups
should achieve targeting via specific recognition by
membrane lectins. After iv or ip injections into rats,
amphiphilic fluorocarbon telomers derived from tris-
(hydroxymethyl)aminomethane (Tris) and bearing per-
fluorocarbon chain were detected in all organs.7 Further-
more, in vitro, these compounds easily cross the plasma
membrane,8 and it is thus reasonable to think that A can
cross cell membranes. We report hereafter the synthesis
of A, and some preliminary results on its trapping ability
and its recognition by specific membrane lectins.
The synthesis of A was performed as reported in
Scheme 2. The glycine derivative 1 was obtained by
reaction of Z-protected glycine with Tris in the presence
of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ)
as a coupling reagent in refluxing ethanol. Acetalization
of two hydroxyl functions of the Tris group was carried
out by treatment with 2,2-dimethoxypropane in acetoni-
trile to yield 1 in 75% yield. The perfluorocarbon chain
was then introduced by reacting 1 with the corresponding
perfluorocarbon isocyanate in the presence of DABCO
followed by hydrolysis of the acetal by Montmorillonite
K10 resin,9 leading to 2 in 81% yield. Glycosylation of
§ Telephone: (33) 4 90 14 44 42. Fax: (33) 4 91 14 44 49. e-mail:
pucci@univ-avignon.fr.
| Telephone: (33) 4 91 28 85 62. Fax: (33) 4 91 28 85 12. e-mail:
chalier@srepir1.univ-mrs.fr.
† Universite´ d’Avignon et des Pays du Vaucluse.
‡ CNRS et Universite´s d’Aix-Marseille I et III.
(1) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J . Arch. Biochem.
Biophys. 1980, 200, 1. Rosen, G. M.; Finkelstein, E. Adv. Free Rad.
Biol. Med. 1985, 1, 345.
(2) Halliwell, B.; Gutteridge, J . M. C. Free Radicals in Biology and
Medicine; Clarendon Press: Oxford, 1989; Chapter 2, p 47. DeGray, J .
A.; Mason, R. P. Biological Spin Trapping. In Electron Spin Resonance;
Atherton, N. M., Davies, M. J ., Gilbert, B. C., Eds.; Athenaeum Press
Ltd.: Cambridge, 1994; Vol. 14, pp 246-301. Thornalley, P. J . Life
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Finkelstein, E.; Russo, A. Free Rad. Biol. Med. 1989, 6, 141.
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Engl. 1988, 27, 113. Dacho, Y.; Okada, S.; Gotto, R. Biochim. Biophys.
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(6) Ouari, O.; Chalier, F.; Pucci, B.; Tordo, P. J . Chem. Soc., Perkin
Trans. 2 1998, 2299.
(7) Maurizis, J . C.; Azim, M.; Rapp, M.; Pucci, B.; Pavia, A. A.;
Madelmont, J . C.; Veyre, A. Xenobiotica 1994, 24, 1183.
(8) Chehade, F.; Maurizis, J . C.; Pucci, B.; Pavia, A. A.; Ollier, M.;
Veyre, A.; Escaig, F.; J eanguillaume, C.; Dennebouy, R.; Slodzian, G.;
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10.1021/jo982343+ CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/24/1999

Glycolipidic Amphiphilic Nitrone as a New Spin Trap
J . Org. Chem., Vol. 64, No. 10, 1999 3555
Sch em e 2a
a
(a) EEDQ, EtOH, reflux, 75%; (b) 2,2-dimethoxypropane, cat. p-TsOH, CH3CN, rt, 85%; (c) C8F17(CH2)2NdCdO, DABCO, toluene,
reflux, 90%; (d) Montmorillonite K10, CH2Cl2, rt, 90%; (e) tetraacetobromogalactose, Hg(CN)2, ultrasound, CH3CN, 65%; (f) H2, Pd-C,
MeOH, 98%; (g) succinic anhydride, pyridine, rt, 94%; (h) ethylene glycol, cat. p-TsOH, toluene, reflux, 90%; (i) AlLiH4, Et2O, 0 °C, 78%;
(j) DCC, HOBT, CH2Cl2, rt, 90%; (k) CH3CHO, cat. p-TsOH, CH2Cl2, rt, 85%; (l) (CH3)3CNHOH, 3 Å molecular sieves, THF, argon, 45 °C,
52%; (m) MeONa, MeOH, Amberlite IRC 50S resin, rt, 95%.
the tris hydroxyl functions by 2,3,4,6-tetra-O-acetyl-R-
D-galactopyranosyl bromide in the presence of mercuric
cyanide under ultrasonic activation led to compound 3.10
Under these conditions, the bisglycosylation was stereo-
selective and afforded the bis â-D-galactopyranoside
derivative 3 in 65% yield after column chromatography.
The Z-amine protecting group was then removed by
hydrogenolysis (10% Pd/C) in MeOH, and the resulting
amine was treated with 2 equiv of succinic anhydride in
pyridine to give 4 in a good yield (92%). On the other
hand, protection of the carbonyl group of 4-cyanobenz-
aldehyde as its ethylene ketal (90%) and reduction of the
cyano group with AlLiH4 in Et2O (78%) afforded the
benzylamine 5 which was then reacted with the tensio-
active moiety 4. The amide 6 was obtained in a good yield
(90%) by the reaction of 4 with 5 in the presence of N,N-
dicyclohexylcarbodiimide (DCC) and a catalytic amount
of 1-hydroxybenzotriazole (HOBT) in dry CH2Cl2 at room
F igu r e 1. Observed (continuous line) and simulated (broken
line) ESR spectra after hydroxyl radical generation with
temperature. Then transacetalization with a large excess
of acetaldehyde regenerated under mild conditions the
benzaldehyde derivative in 85%. Condensation of the
aldehyde with N-tert-butylhydroxylamine in dry THF
introduced the nitrone function.11 Owing to the possible
degradation of hydroxylamine by oxidation followed by
free radical processes, the reaction was carried out in the
H2O2-FeSO4-EDTA system in phosphate buffer at pH 7 in
the presence of nitrone A and (a) 2 mM sodium formate, (b)
10% methanol.
dark and under an inert atmosphere. Purification on
silica gel and on Sephadex LH-20 resin, eluting with
EtOH:CH2Cl2 (1:1), yielded the pure nitrone as a white
foam (55%) without residual ESR signal. Removal of the
O-acetyl protecting groups using MeONa/MeOH followed
by purification on Sephadex G-25 resin (distilled water
as eluant) afforded the â-D-galactopyranoside nitrone A.
(10) Polidori, A.; Pucci, B.; Maurizis, J . C.; Pavia, A. A. New. J .
Chem. 1994, 18, 839.
(11) Hinton, R. D.; J anzen, E. G. J . Org. Chem. 1992, 57, 2446.

3556 J . Org. Chem., Vol. 64, No. 10, 1999
Ouari et al.
As expected, compound A shows a surfactant behavior,
with a critical micellar concentration (cmc) of 1.6 10-5
mol/L and a surface tension of 28 mN/m.
flocculation processes involving lectins.6,12 The yeast used
was Kluyveromyces bulgaricus and involved the galactose
specific lectin Kb CWL1. The affinity of nitrone A for Kb
CWL1 was higher than that of free galactose. This
recognition feature could lead to specific spin trap
distribution in biological systems, and work is now in
progress to determine the distribution and localization
of nitrone A in cell culture.
Preliminary spin trapping experiments with A were
performed, and the shape of the ESR spectra was
strongly dependent upon the nature of the trapped
•
•
radical. The radicals COO-,Na+ or CH2OH were gener-
ated in phosphate buffer (pH 7) by a Fenton system with
sodium formate or methanol, respectively, in the presence
of A (100 mM). The resulting ESR spectra are shown in
Figure 1. The use of 13C-labeled sodium formate or
methanol unambiguously supported the nature of the
A-CO2-,Na+ and A-CH2OH spin adducts. The spectra
were satisfactorily simulated by assuming that they
correspond to the superimposition of the spectra of a spin
adduct differently immobilized in two distinct environ-
ments. When dioxane was added to break the organiza-
tion of the amphiphilic nitrone A, the percentage of the
spectrum corresponding to the less immobilized spin
adduct increased significantly.
Ack n ow led gm en t. We wish to thank Pr. Roger
Bonaly and Dr. J oel Coulon from the University of
Nancy for their collaboration.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures including synthesis and characterization of all new
compounds reported herein. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O982343+
To determine the lectin recognition ability of A, its
inhibiting property was measured by using classical yeast
(12) Al Mahmood, S.; Colin, S.; Bonaly, R. J . Biol. Chem. 1991, 266,
20882.