A new glucosamine-containing amphiphilic spin probe

Article abstract of DOI:10.1016/j.tetlet.2008.11.075

A new, nonionic amphiphilic spin probe for investigating the extracellular matrix close to the cell membrane by EPR spectroscopy has been synthesized and characterized. A pyrrolidine type nitroxide spin-label has been introduced to the third position of a

Full text of DOI:10.1016/j.tetlet.2008.11.075

Tetrahedron Letters 50 (2009) 567–569
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Tetrahedron Letters
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A new glucosamine-containing amphiphilic spin probe
a,b
Janez Mravljak a, , Slavko Pecar
*
ˇ
a
ˇ
Faculty of Pharmacy, University of Ljubljana, Aškerceva 7, 1000 Ljubljana, Slovenia
b
ˇ
Institute Jozef Stefan, Jamova 39, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
A new, nonionic amphiphilic spin probe for investigating the extracellular matrix close to the cell mem-
brane by EPR spectroscopy has been synthesized and characterized. A pyrrolidine type nitroxide spin-
label has been introduced to the third position of a nonionic sugar polar head (glucosamine) bonded
to a lipophilic stearic acid acyl chain anchor. The compound is soluble in polar organic solvents such
as ethanol and chloroform, but is sparingly soluble in water.
Received 24 September 2008
Revised 8 November 2008
Accepted 19 November 2008
Available online 24 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Spin probes
Nitroxide free radicals
Glucosamine
Glycocalyx
The extracellular space in tissues is filled with an intricate net-
work of macromolecules constituting the extracellular matrix. It is
composed of a variety of proteins and polysaccharides that are se-
creted locally and assembled into an organised meshwork in close
association with the surface (glycocalyx) of the cell that produced
them. Besides its scaffold-forming function that stabilizes the
structure of the tissue, it has a far more active and complex role
in regulating the behaviour and function of the cells in contact
with it. Despite the rapid progress in characterizing its major com-
ponents, our understanding of its organization is still incomplete.1
EPR has proved to be a powerful method for monitoring the bio-
logical characteristics of plasma membranes, their interactions
with spin-labelled compounds and the dynamics of the latter.2
The fine structure of the EPR spectra provides information on the
dynamics, polarity, pH, and structural and redox properties of the
environment of the spin-labelled molecule.3 The successful appli-
cation of spin-labelled compounds depends strongly on properly
designed spin probes, which are mostly stable free radicals of the
nitroxide type.4
The ideal spin probe for investigating the extracellular matrix
close to the cell surface should be an amphiphilic molecule that
can be easily and well anchored into the phospholipid bilayer, with
negligible surfactant effects on the integrity of the cell membrane.
It should therefore contain a single alkyl chain, but be sufficiently
water soluble to pass through the water phase from the film on the
test tube wall to the cell surface in the labelling procedure.
Charged groups in the polar head should be avoided to reduce their
possible haemolytic effects. The nitroxide group-containing moiety
should sense the extracellular matrix, therefore it should be posi-
tioned as high as possible on the polar head, with a predominantly
perpendicular orientation to the membrane surface (along the z
axis) of the 2pp orbital containing the unpaired electron, to ensure
the highest possible sensitivity of the probe.
The design of the new spin probe 8 reported here was based on
an amphiphilic molecule composed of three components: a stea-
royl alkyl chain as a flexible lipophilic anchor, a glucosamine sugar
moiety as a nonionic polar bulky head and a pyrrolidine type nitr-
oxide free radical as the spin-label. The bulky polar head bearing
the nitroxide spin-label is the more rigid part of the molecule
and is much larger in cross-section than the flexible alkyl chain.
To synthesize the desired spin probe, we used the synthetic
strategy presented in Scheme 1. In the first step, the lipophilic alkyl
chain was introduced to the starting sugar,
a-D-glucosamine
hydrochloride 1, by selective N-acylation with stearoyl chloride
in dioxane/water mixture at ambient temperature.5 In the two fol-
lowing steps, 1-O-benzyl and 40,60-benzylidene protecting groups
were introduced according to known procedures, leaving the
30-hydroxy group of 4 free for a further reaction step.6 Fischer gly-
cosylation of 2 with benzyl alcohol over a prolonged reaction time
in refluxing benzene gave predominantly the a
-anomer of 3.7
The dihydropyrrole-type nitroxide spin-label was introduced by
a Williamson reaction, using bromo-derivative 5.8 The reaction
was carried out in anhydrous dioxane with sodium hydride as
the base and 15-crown-5 as a catalyst to give 69 in good yield.10
Next, the benzylidene and benzyl protecting groups were removed
by hydrogenolysis in acetic acid/methanol mixture. Also in this
reaction step, the nitroxide group was reduced to an amine and,
surprisingly, the double bond of the dihydropyrrole ring was re-
duced, affording a new chiral centre. The resulting secondary
* Corresponding author. Tel.: +386 1 47 69 500; fax: +386 1 42 58 031.
E-mail address: janez.mravljak@ffa.uni-lj.si (J. Mravljak).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.11.075

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J. Mravljak, S. Pecar / Tetrahedron Letters 50 (2009) 567–569
568
OH
OH
OH
Ph
O
O
O
O
O
O
b
HO
HO
c
a
HO
HO
HO
HO
HO
OH
NH
NH
NH
O
H3N
Ph
O
Ph
OH
Cl
O
O
O
C17H35
C17H35
C17H35
1
2
3
4
OH
O
OH
O
Br
Ph
O
O
O
O
O
HO
HO
d
f
e
4
O
O
O
+
N
O
OH
OH
NH
NH
NH
Ph
O
O
C17H35
C17H35
C17H35
N
N
5
HN
O
O
6
7
8
Scheme 1. Reaction and conditions: (a) H3C(CH2)16COCl, NaHCO3, dioxane/water, rt, 6 h, 94%; (b) BnOH, PTSA, benzene, reflux, 12 h, 65%; (c) PhCHO, (EtO)3CH, PTSA, DMF,
dioxane, rt, 12 h, 74%; (d) NaH, 15-crown-5, dioxane, 50 °C, 40 h, 79%; (e) H2, Pd/C, AcOH, MeOH, 64%; (f) H2O2, Na2WO4ꢀ2H2O, Na-EDTA, MeOH, water, rt, 48 h, 56%.
amine 7 was oxidized to nitroxide 811 using hydrogen peroxide in
the presence of sodium wolframate as catalyst.12 HPLC analysis
revealed that the final compound 8 was a mixture composed of
four diastereoisomers—two pairs of mutarotamers that can inter-
convert in solution.
a
b
c
The new spin probe 8 was found to be sufficiently soluble in
ethanol and only sparingly soluble in water (Fig. 1). Its surfactant
properties were estimated from its haemolytic activity.11 In con-
trast to some other amphipathic spin probes possessing charged
groups on their polar heads, compound 8 exerts practically no hae-
molytic activity, even at the high molar ratio of one molecule of 8
per 100 lipid molecules in membranes of erythrocytes.13
In order to test the suitability of the new spin probe for extra-
cellular matrix investigation in cell cultures, it was incorporated
into liposomes composed of gangliosides (type III) from bovine
brain and lecithins (Emulmetic 320).14 The X-band spectra of com-
pound 8 recorded at different temperatures (Fig. 2) show signifi-
cant immobilization of the spin probe, consistent with the
position of the spin probe in the sugar chain region of gangliosides
close to the lipid-water interface.
d
mT
Figure 2. X-band EPR spectra of 8 incorporated into liposomes15 recorded at
different temperatures: (a) 47 °C, (b) 37 °C, (c) 27 °C, and (d) 20 °C.
ary EPR measurements on liposomes have confirmed its potential
for characterizing the order and dynamic properties of the extra-
cellular matrix close to the cell surface. The properties of cancer
cell surfaces are now under investigation, and EPR studies are ex-
pected to provide useful information on the glycocalyx properties
of relevant biological samples.
In conclusion, we have synthesized a new, nonionic amphiphilic
spin probe for investigating the extracellular glycocalyx. Prelimin-
Acknowledgements
a
b
This work was supported by the Ministry of Higher Education,
Science and Technology of the Republic of Slovenia. The authors
thank Professor Roger Pain for critical reading of the manuscript.
References and notes
1. Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular
Biology of the Cell; Garland Science, a member of the Taylor & Francis Group:
New York, 2008.
c
2. Griffith, O. H.; Jost, P. C. In Spin Labeling I, Theory and Applications; Berliner, L. J.,
Ed.; Academic Press: New York, 1976; pp 453–523; McConnell, H. M. In Spin
Labeling I, Theory and Applications; Berliner, L. J., Ed.; Academic Press: New York,
1976; pp 525–560.
mT
3. Ge, M.; Freed, J. H. Biophys. J. 1998, 74, 910–917; Hemminga, M. A.; Berliner, L. J.
ESR Spectroscopy in Membrane Biophysics; Springer, 2007.
4. Likhtenshtein, G. I. In Nitroxides, Likhtenstein, G. I., Yamauchi, J., Nakatsuji, S.,
Smirnov, A. I., Tamura, R., Eds.; Wiley-VCH: Weinheim, 2008; pp 205–238.
5. Fieser, M.; Fieser, L. F.; Toromanoff, E.; Hirata, Y.; Heymann, H.; Tefft, M.;
Bhattacharya, S. J. Am. Chem. Soc. 1956, 78, 2825–2832.
Figure 1. X-band EPR spectra of
saturated solution of 8, estimated solubility, 11
presence of atmospheric O2 and (c) de-aerated ethanol solution (1 mM), all at room
temperature.
8
in (a) phosphate buffer solution (pH 7.4,
M), (b) ethanol (1 mM) in the
l

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J. Mravljak, S. Pecar / Tetrahedron Letters 50 (2009) 567–569
569
6. Gross, P.; Rimpler, M. Liebigs Ann. Chem. 1986, 1, 37–45.
10. Aspinall, H. C.; Greeves, N.; Lee, W.-M.; McIver, E. G.; Smith, P. M. Tetrahedron
Lett. 1997, 38, 4679–4682.
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7. Babic, A.; Pecar, S. Synth. Commun. 2008, 38, 3052–3061.
8. Hankovszky, H. O.; Hideg, K.; Lex, L. Synthesis 1980, 11, 914–916.
9. Benzyl-4,6-O-benzylidene-2-deoxy-2-stearoylamido-3-O-(1-oxyl-2,2,5,5-tetrame-
11. 2-Deoxy-2-stearoylamido-3-O-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyr-
rol-3-yl)methyl-a-D-glucopyranoside (8): Yellow crystals, mp 102–110 °C. IR
thyl-2,5-dihydro-1H-pyrrol-3-yl)methyl-
crystals, mp 110–112 °C. IR (KBr, cmꢁ1): 3315, 2924, 2854, 1646, 1540,
1456, 1373, 1079. 1H NMR (CDCl3, 300 MHz):
(ppm) 0.88 (t, 3H,
a
-
D-glucopyranoside
(6):
Yellow
(KBr, cmꢁ1): 3310, 2924, 2852, 1642, 1560, 1408, 1262, 1124, 1048. LRMS
(EI), m/z = 599 (M)+. HRMS (ESI), m/z calcd for C33H63N2O7 599.4635 (M)+,
found 599.4652. EPR: aN (ethanol) = 1.513 mT. Calcd C33H63N2O7ꢀ0.5 NH3: C,
65.15; H, 10.69; N, 5.76. Found: C, 65.28; H, 10.69; N, 5.76. HPLC: Column C18
d
J = 6.5 Hz, CH3), 1.15, 1.17 (2s, 12H, cis-, trans-2,5-CH3), 1.25 (br s, 30H,
CH2), 2.12 (t, 2H, J = 7.5 Hz, CO–CH2), 3.60–3.79 (m, 2H, H-6, H-4), 3.86–
3.93 (m, 1H, H-5), 4.05 (app t, 1H, J = 13.7 Hz, H-3), 4.25 (dd, 1H, J = 4.4 Hz,
J = 10.0 Hz, H-6), 4.35–4.38 (m, 1H, H-2), 4.45 (d, 1H, Jgem = 11.8 Hz,
CH2aPh), 4.71 (d, 1H, Jgem = 11.8 Hz, CH2bPh), 4.93 (d, 1H, J = 3.6 Hz, H-1),
5.25 (s, 2H, C–CH2–O), 5.47 (s, 1H, C@CH), 5.55 (s, 1H, CH–Ph), 5.70 (d, 1H,
J = 8.8 Hz, NH),7.18 (s, 1H, N–OH), 7.32–7.48 (m, 10H, H-Ar). 13C NMR
(CDCl3, 75 MHz): d (ppm) 172.47, 141.47, 137.14, 136.76, 129.59, 128.87,
128.29, 128.14, 128.08, 127.89, 125.91, 125.77, 100.98, 97.69, 82.61, 77.19,
69.92, 69.54, 68.92, 68.81, 67.59, 62.96, 52.16, 36.79, 31.80, 29.58, 29.54,
29.40, 29.24, 25.68, 25.58, 24.61, 24.48, 22.57, 14.00. LRMS (EI), m/z = 775.7
(M)+. HRMS (ESI), m/z calcd. for C47H71N2O7 775.5261 (M)+, found 775.5273.
EPR: aN (ethanol) = 1.55 mT. Calcd for C47H71N2O7: C, 72.74; H, 9.22; N,
3.61. Found: C, 73.14; H, 9.42; N, 3.77. NMR spectra were obtained after
reduction of nitroxides to hydroxylamines with phenyl hydrazine
(1.2 equiv) under an argon atmosphere.
Synergy 10
1.0 mL/min; injection volume: 20
l
; mobile phase: gradient 60–90% acetonitrile, water; flow rate
L; retention times: 18.88 min (3.84%),
l
19.44 min (14.22%), 20.00 min (23.36%) and 20.72 min (58.59%) at 210 nm.
12. Rozantsev, E. G. Free Nitroxyl Radicals; Plenum Press: New York, 1970; Plessas,
N. R.; Goldstein, I. J. Carbohydr. Res. 1981, 89, 211–220.
13. Stensrud, G.; Passi, S.; Larsen, T.; Sandset, P. M.; Smistad, G.; Mönkkönen, J.;
Karlsen, J. Int. J. Pharm. 1999, 178, 33–46.
ˇ
14. Mravljak, J.; Zeisig, R.; Pecar, S. J. Med. Chem. 2005, 40, 6393–6399.
15. Multilamellar liposomes were prepared by the lipid film method: gangliosides
(type III) from bovine brain (5.2
and 8 (0.2 mol) were dissolved in 3 mL of chloroform and 1 mL of methanol.
The organic solvents were evaporated on a rotary evaporator in a glass flask,
forming a lipid film and then 180 of a 0.99 M solution of sucrose in
lmol), lecithins (Emulmetic 320) (21.6 lmol)
l
l
L
phosphate buffer saline (PBS, pH 7.4) was added to the flask. The suspension
was hand-shaken and sonicated for 15 min.