Synthesis, physical-chemical and biological properties of amphiphilic amino acid conjugates of nitroxides

Article abstract of DOI:10.1039/c0nj00024h

Due to the dual property of synthetic nitroxide compounds to either act as probe or antioxidant, efforts toward their selective targeting using specific ligands have been extensively explored. Herein, we report the synthesis of novel amphiphilic nitroxides in which the nitroxyl group is grafted onto an amphiphilic carrier comprising a lactobionamide polar group, a non-polar alkyl chain and an amino acid as scaffold. Piperidine and pyrrolidine nitroxides such as 4-amino-TEMPO (4-AT) and 3-carboxyproxyl (3-CP), respectively, were grafted onto the amphiphilic carriers. To further investigate the effect of the nature of the chain on the physical-chemical and biological properties of nitroxides, hydrogenated or perfluorinated alkyl chains were used. The self-aggregation properties in aqueous media of these surfactant-like nitroxides were confirmed by dynamic light scattering (DLS) as well as electron paramagnetic resonance (EPR) spectroscopy, and were correlated with their respective lipophilicity. The effect of the carrier groups on the electrochemical property of nitroxides was investigated using cyclic voltammetry, and the rates of reduction using ascorbate as reducing agent were measured. Finally, their cytoprotective property against toxic concentrations of hydrogen peroxide using bovine aortic endothelial cells was also investigated.

Full text of DOI:10.1039/c0nj00024h

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www.rsc.org/njc | New Journal of Chemistry
Synthesis, physical-chemical and biological properties of amphiphilic
amino acid conjugates of nitroxidesw
a
a
b
c
´
Gregory Durand,* Fanny Choteau, Robert A. Prosak, Antal Rockenbauer,
Frederick A. Villamena*bd and Bernard Puccia
Received (in Montpellier, France) 13th January 2010, Accepted 18th March 2010
DOI: 10.1039/c0nj00024h
Due to the dual property of synthetic nitroxide compounds to either act as probe or antioxidant,
efforts toward their selective targeting using specific ligands have been extensively explored.
Herein, we report the synthesis of novel amphiphilic nitroxides in which the nitroxyl group is
grafted onto an amphiphilic carrier comprising a lactobionamide polar group, a non-polar
alkyl chain and an amino acid as scaffold. Piperidine and pyrrolidine nitroxides such as
4-amino-TEMPO (4-AT) and 3-carboxyproxyl (3-CP), respectively, were grafted onto the
amphiphilic carriers. To further investigate the effect of the nature of the chain on the physical-
chemical and biological properties of nitroxides, hydrogenated or perfluorinated alkyl chains were
used. The self-aggregation properties in aqueous media of these surfactant-like nitroxides were
confirmed by dynamic light scattering (DLS) as well as electron paramagnetic resonance (EPR)
spectroscopy, and were correlated with their respective lipophilicity. The effect of the carrier
groups on the electrochemical property of nitroxides was investigated using cyclic voltammetry,
and the rates of reduction using ascorbate as reducing agent were measured. Finally, their
cytoprotective property against toxic concentrations of hydrogen peroxide using bovine aortic
endothelial cells was also investigated.
they exhibit protective properties against toxicity induced by
Introduction
reactive oxygen species in cell cultures8 and whole animals.7
However, the mechanism of the antioxidant property of
nitroxides is poorly understood, but is believed to be due to
their ability to catalytically remove superoxide radical anion
Over the past decades, nitroxides have been extensively
employed as building blocks for the development of molecular
magnetic materials1 and as modulators of polymerization
processes.2 Using electron paramagnetic resonance (EPR)
spectroscopy, nitroxides have also been employed as spin
probes for the investigation of membrane dynamics,3 metabolism
and oxygenation,4 pH and redox status in cellular systems.5
Moreover, nitroxides have been used as contrast agents for
magnetic resonance imaging.6 The therapeutic properties of
nitroxides7 have been gaining attention over recent years since
(O2 ꢁ) via an SOD-like dismutation mechanism, and therefore
ꢀ
may offer cellular protection from the direct and indirect
ꢁ 9
ꢀ
toxicity of O2
. The SOD-mimetic property of nitroxides
could proceed via ꢀtwo mechanisms: (1) through reduction of
the nitroxide by O2 ꢁ to its hydroxylamine form and subsequent
ꢁ
ꢀ
reoxidation by another molecule of O2
to regenerate
the nitroxide; (2) or via oxidation of the nitroxide to the
oxammonium cation, and subsꢁeq1u0ent reduction to the nitroxide
a
ꢀ
`
´
Laboratoire de Chimie BioOrganique et des Systemes Moleculaires
by another molecule of O2
.
Also, worth noting is the
´
´
Vectoriels, Faculte des Sciences, Universite d’Avignon et des Pays de
Vaucluse, 33 Rue Louis Pasteur, 84000 Avignon, France.
E-mail: gregory.durand@univ-avignon.fr; Fax: +33 4 9014-4441;
Tel: +33 4 9014-4445
ability of nitroxides to react with carbon-, sulfur- and oxygen-
centered radicals10 as well as the ability to oxidize iron(II)9 or
reduce ferryl heme,11 thereby contributing to their antioxidant
efficacy. Moreover, nitroxides have been shown to prevent
DNA damage induced by transition metal ions12 or ionizing
radiation,13 as well as inhibit myeloperoxidase-mediated
hypochlorous acid production.14
b Department of Pharmacology, The Davis Heart and Lung Research
Institute, College of Medicine, The Ohio State University, Columbus,
OH 43210, USA. E-mail: frederick.villamena@osumc.edu;
Fax: +1 614-688-0999; Tel: +1 614-292-8215
c Chemical Research Center, Institute of Structural Chemistry,
H-1025 Budapest, Pusztaszeri 59, Hungary
Due to the nitroxide dual property to either act as probe or
antioxidant, the selective targeting of nitroxides using specific
ligands has been an active field of investigation during the last
decade. For example, the triphenylphosphonium group was
conjugated to pyrrolidine15 and piperidine nitroxides16,17 and
has been employed as a mitochondria-target molecule. The
accumulation of these targeted nitroxides in the mitochondria
was demonstrated using EPR spectroscopy as well as by using
a selective triphenylphosphonium electrode. An alternative
strategy was employed through the use of gramicidin analogues
d Center for Biomedical EPR Spectroscopy and Imaging,
The Davis Heart and Lung Research Institute, College of Medicine,
The Ohio State University, Columbus, OH 43210, USA
w Electronic supplementary information (ESI) available: Materials,
general procedures and instrumentation for the synthesis, experimental
procedures for 10, 11, 13 and 14; materials and general procedures for
cell culturing; 1H and 13C spectra of 1, 2, 5 and 6; MS spectra of 2, 3, 6,
7, 8, 10 and 13; HRMS spectra of 4, 11 and 14; concentration
dependence of nitrogen coupling and linewidth of 3-CP and 4-AT in
PBS; X-band EPR spectra of clustered and non-clustered 4 and 11 in
PBS; radical distribution as function of the concentration of 4, 7, 11
and 14; nitroxide stability data. See DOI: 10.1039/c0nj00024h
ꢂc
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that ensure favorable mitochondrial targeting.18 On the other
hand, membrane-targeted nitroxides in which a lipophilic tail
serves as an anchor were synthesized19,20 and were employed
to investigate oxidative stress occurring within the cell.
Several amphiphilic antioxidants have been developed in
our group as potential therapeutic agents with the expectation
that amphiphilic compounds possessing both hydrophilic and
lipophilic groups may exhibit improved cellular permeability.21–23
Amphiphilic antioxidants are comprised of a hydrophilic polar
head which provides water solubility while a hydrophobic tail
ensures sufficient lipophilicity to pass through cell membranes.
Amphiphilic a-phenyl-N-tert-butyl nitrone derivatives have
shown enhanced protective properties against reactive oxygen
species-mediated toxicity in in vitro,22,24 ex vivo25 and in vivo
models,26,27 compared to unconjugated nitrones, further
validating the effect of amphiphilic group on their protective
properties. To extend the concept of amphiphile conjugation
to other antioxidants, fluorinated amphiphilic amino acids
have been developed.23,28 Lysine and aspartic acid were used
as scaffolds upon which a hydrophilic lactobionamide group
and a lipophilic fluorinated chain were grafted to the primary
functional groups while the antioxidants were grafted to the
side chain. In particular, we have recently reported the synthesis
and the cytoprotective properties of the first fluorinated
amphiphilic cyclic nitrone derivative.29 Indeed, while amphiphilic
compounds endowed with a hydrocarbon chain can destabilize
cell membranes when used above their critical micellar
concentration (cmc), fluorinated amphiphilic analogues are
devoid of such detergent properties.30 However, our previous
findings with amphiphilic nitrones show conflicting results
where the LPBNAH which is conjugated to hydrogenated
carrier shows greater biological activity compared to its
fluorinated analogues,24,27 suggesting other factors are involved
for their bioactivity.
Fig.
1
Structures of hydrogenated and fluorinated amphiphilic
carriers grafted on various types of nitroxides.
against toxic concentrations of hydrogen peroxide using
bovine aortic endothelial cells was investigated.
Results and discussion
Synthesis
The synthetic strategy for the amphiphilic amino acid nitroxides
is based on three key steps: (i) introduction of an alkyl chain
on a protected amino acid, (ii) condensation of a lactobionamide
polar group on the hydrophobic amino acid derivative, and
(iii) conjugation of the nitroxide on the amino acid side chain.
Condensation of octylamine with Na-(9-fluorenylmethyl-
oxycarbonyl)-L-aspartic acid-b-tert-butyl ester in the presence
of dicyclohexylcarbodiimide (DCC)/hydroxybenzotriazole
(HOBt) gave compound 1 with 92% yield (Scheme 1). N-Fmoc
deprotection was achieved under basic condition in the
presence of diethylamine in acetonitrile, and the resulting
amino compound was grafted on lactobionolactone, prepared
according to our previously described procedure.23 Acetylation
of the crude mixture gave compound 2 with 39% yield in three
steps. The tert-butoxycarbonyl protective group was removed
under acidic conditions in the presence of trifluoroacetic acid
in dichloromethane, and condensation of 4-AT on the resulting
carboxylic acid derivative was achieved in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/HOBt
to give, after purification, compound 3 with 46% yield in
With the goal to prove whether the grafting of a nitroxide
with an amphiphilic carrier may affect its bioavailability and
bioactivity, we present in this work the synthesis of novel
hydrogenated-octyl chain amphiphilic amino acid-based cyclic
nitroxides (Fig. 1) designed to provide amphiphilic character
to nitroxides without inducing cellular toxicity. Indeed, sugar-
based octyl surfactants exhibit relatively high cmc (around
20 mM) so no toxicity should be detected if used below their
cmc. Piperidine and pyrrolidine nitroxides using 4-amino-
TEMPO (4-AT) and 3-carboxyproxyl (3-CP), respectively,
were grafted to the amphiphilic carriers. To investigate the
effect of the nature of the chain on the physical-chemical and
biological properties of nitroxides, fluorinated amphiphilic
amino acid-based nitroxides were also prepared. The self-
aggregation properties in aqueous media of these surfactant-like
nitroxides were confirmed by dynamic light scattering (DLS)
as well as electron paramagnetic resonance (EPR) spectro-
scopy and were correlated with their respective lipophilicity.
The effect of the carrier groups on the electrochemical properties
of the nitroxides was also investigated using cyclic voltammetry,
and the rates of reduction of the aminoxyl group using
ascorbate as reducing agent were measured. Moreover, by
comparing the physical-chemical data of TEMPO, 4-AT and
its amido derivative 4-AcT, (Fig. 1) we pointed out the effect
of the substituent. Finally, their cytoprotective property
two steps. Finally, Zemple
´
n de-O-acetylation gave the
amphiphilic nitroxide 4.
Condensation of octylamine on Na-tert-butyloxycarbonyl-
Ne-benzyloxycarbonyl-L-lysine in the presence of DCC/HOBt
gave 5 with 74% yield (Scheme 2). After N-Boc group removal
under acidic conditions, the resulting lysine amino intermediate
was grafted onto lactobionolactone, followed by acetylation of
the hydroxyl groups to give compound 6 in 50% yield in three
steps. Benzyloxycarbonyl group removal was achieved by
catalytic hydrogenolysis, and the resulting amino compound
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The fluorinated carriers were prepared as previously
described by using 1H,1H,2H,2H-perfluorooctylamine.23
Compounds 11 and 14 were prepared following the same
procedure employed for 4 and 8, respectively (Scheme 3).
Deprotection of 9 under acidic conditions followed by grafting
of the 4-AT in the presence of EDC/HOBt gave compound 10
in two steps in 60% yield. The benzyloxycarbonyl group of 12
was removed by catalytic hydrogenolysis, and the resulting
amino compound was added to 3-CP in the presence of
EDC/HOBt to give compound 13 in 28% yield. Finally,
Zemplen de-O-acetylation of 10 and 13 gave the amphiphilic
´
nitroxides 11 and 14, respectively.
The hydrogenated carriers 2 and 6 were fully characterized
by NMR experiments as well as by mass spectrometry, while
only the latter characterization was performed on the
conjugated compounds 4, 8, 11 and 14 (see ESIw for details).
As shown in Fig. S3 and S6, the 1H NMR spectra of
the carriers show the presence of relevant moieties such as the
acetyl groups of the polar head at 1.95–2.25 ppm as well as the
alkyl chain, with a methyl group at 0.9 ppm and five methylene
groups at 1.20–1.40 ppm. 13C NMR and DEPT experiments
allowed us to identify the resonance peaks of the methines and
methylenes of the lactobionamide group as well as those of the
amino acids. For instance, while the methine group of the acid
aspartic carrier is at 49.0 ppm that of the lysine carrier is at
52.7 ppm. The carbonyl carbons of the ester and amide
bonds were also observed on the 13C spectra, confirming the
structures of compounds 2 and 6.
Scheme 1 Synthesis of 4-AT aspartic acid derivative 4. Reagents:
(a) octylamine, DCC/HOBt, CH2Cl2; (b) DEA–CH3CN 1 : 9 (v/v);
(c) lactobionic acid, TEA, methoxyethanol; (d) Ac2O–pyridine 1 : 1
(v/v); (e) TFA–CH2Cl2 4 : 6 (v/v); (f) 4-AT, EDC/HOBt, CH2Cl2;
(g) MeONa, methanol.
For simplicity in naming, the nitroxides are denoted by
abbreviated names indicating their chemical structures. The
first letter L stands for lactobionamide group, followed by a
second letter indicating the nature of the amino acid, with
A for aspartic acid and L for lysine, while the third
letter indicates the nature of the alkyl chain, with H for the
hydrogenated and F for the fluorinated derivative. For
example, LAH-4-AT denotes the hydrogenated aspartic acid
derivative bearing the 4-AT nitroxide. A similar nomenclature
was used for the carrier devoid of nitroxide moiety.
Partition coefficients
All the amphiphilic nitroxides obtained are highly soluble in
water at 25 mM; however, solutions of fluorinated derivatives
11 and 14 were slightly viscous at this concentration. Their
relative lipophilicities (logk0W) were measured by the HPLC
technique as previously used,22 and the data are shown in
Table 1. The values for the parent compounds (i.e., 4-AT and
3-CP) are in good agreement with those obtained by calculation
(Table 1). In spite of the high solubility of the conjugates in
water, they were found to be more lipophilic than the parent
nitroxide compounds. The fluorinated analogues LLF-3-CP
and LAF-4-AT exhibited higher lipophilicities than their
hydrogenated analogues LLH-3-CP and LAH-4-AT, as
demonstrated by their higher logk0W values (Table 1). It
appears that the polarity difference between LLH-3-CP and
LAH-4-AT is less pronounced than that of the parent
compounds. The same observation is true with the fluorinated
derivatives. This demonstrates that the lipophilicity of
amphiphilic nitroxides is mainly determined by the nature of
Scheme 2 Synthesis of 3-CP lysine derivative 8. Reagents: (a)
octylamine, DCC/HOBt, CH2Cl2; (b) TFA–CH2Cl2 3 : 7 (v/v); (c)
lactobionic acid, TEA, methoxyethanol; (d) Ac2O–pyridine 1 : 1
(v/v); (e) H2, 7 bar, Pd/C, ethanol–AcOH 99 : 1 (v/v); (f) 3-CP,
EDC/HOBt, CH2Cl2; (g) MeONa, methanol.
was added to 3-CP in the presence of EDC/HOBt to give
compound 7 in 32% yield after purification from 6. Finally,
de-O-acetylation of 7 led to compound 8.
ꢂc
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Scheme 3 Synthesis of fluorinated 4-AT and 3-CP derivatives 11 and 14. Reagents: (a) TFA–CH2Cl2 4 : 6 (v/v); (b) 4-AT, EDC/HOBt, CH2Cl2;
(c) MeONa, methanol; (d) H2, 7 bar, Pd/C, ethanol–AcOH 99 : 1 (v/v); (e) 3-CP, EDC/HOBt, CH2Cl2.
Table 1 Partition coefficients, oxidation potential and relative reduction rate constant of the nitroxides
E1/2 (ox)e,f (mV)
DEp (ox)e,g (mV)
Reduction ratei
a
Nitroxide
logk0 (RP-HPLC)
C logP
W
4-AT
1.1
3.6
5.1
1.4
3.7
5.2
1.05c, 0.9d
—
—
612 ꢃ 7
532 ꢃ 14
545 ꢃ 11
288h
64 ꢃ 2
58 ꢃ 2
60 ꢃ 3
63h
—
b
LAH-4-AT
LAF-4-AT
3-CP
LLH-3-CP
LLF-3-CP
4-AcT
117.0
114.1
1.0
3.2
3.0
b
1.61c, 1.4d
—
b
582h
62h
b
—
0.64c, 1.2d
3.04c, 2.6d
664h
421 ꢃ 5
402 ꢃ 5
d
60h
b
—
—
65 ꢃ 3
106.7
23.1
b
TEMPO
66 ꢃ 4
a
b
c
This work. Not determined. Data using Chemdraw 8.0 software. Data for the hydroxylamine form of the nitroxides using the online
calculator molecular properties on the Molinspirations Web Site (www.molinspiration.com/cgi-bin/properties) as reported by Jiang et al.31 e 0.15 M
NaCl in water, glassy carbon electrode, sweep rate 0.1 V sꢁ1. Average of 4 measurements unless otherwise indicated. E1/2 = (Epc + Epa)/2.
f
g
h
. Average of 2 measurements. In the presence of 5 mM ascorbate and determined by EPR. Data are average of two measurements
i
DEp = Epc ꢁ Epa
relative to 3-CP.
the carrier, with the lysine derivatives being slightly more
lipophilic than the aspartic acid derivatives, which is in perfect
agreement with our previous results with PBN derivatives.23
Therefore, introduction of an amphiphilic character to nitroxides
increases their lipophilicity but maintains water solubility.
Dynamic light scattering
Using dynamic light scattering (DLS) method, the self-
aggregation properties of the amphiphilic nitroxides in water
was studied. As shown in Fig. 2 and Table 2, all the amphi-
philic nitroxides form aggregates with a volume distribution
that is strongly dependent on their structure and concentration.
At 10 mM, both LAF-4-AT and LLF-3-CP self-organize into
well-defined monodisperse particles with B5.5–6 nm diameter.
The amphiphilic PBN derivatives, LLF-PBN and LAF-PBN,
were shown to form micelles at concentrations of 0.05 and
0.35 mM.23 These previous findings, and the similarities in the
structures between the fluorinated nitroxides and PBN
derivatives, suggest that the aggregates formed by LLF-3-CP
and LAF-4-AT at 10 mM are likely spherical micelles, as
previously observed for glucose-based fluorinated surfactants.32
Fig. 2 Particle size distribution by volume of LAF-4-AT, LLF-3-CP,
LAH-4-AT and LLH-3-CP at 10 mM and 25 1C. The graphs report
are an average of 8–10 measurements.
Using 20 mM solution of LAH-4-AT, two peaks of B20 and
B110 nm diameters were observed, indicating the coexistence
of two types of particles in almost equal volume distribution,
with the latter being very likely polydisperse aggregates. On
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Table 2 Aggregate sizes of amphiphilic nitroxides in aqueous solution
LAH-4-AT
LAF-4-AT
LLH-3-CP
LLF-3-CP
a
a
a
a
DH
HHWb
DH
HHWb
DH
HHWb
32.5
DH
HHWb
Concentration
20 mM
c
c
—
c
—
c
19.0 (59%)
110.9 (41%)
20.0 (51%)
105.1 (49%)
18.7 (46%)
109.0 (53%)
21.0 (27%)
108.1 (71%)
5.1
67.8
5.8
118.7
6.4
73.7
5.4
—
29.2
30.7
29.6
33.6
—
10 mM
5 mM
1 mM
5.5
5.6
1.2
1.2
35.8
37.3
44.1
6.0
5.9
5.8
1.3
1.3
1.2
21.0 (38%)
133.6 (57%)
6.0
136.2
71.6
a
DH: hydrodynamic diameter of particles of the main peak in nm. The values reported are average of 8–10 measurements. The percentage volume
b
distribution is higher than 98% otherwise mentioned in bracket. HHW, the width of the peak at half-height in nm, which is an indication of the
c
degree of polydispersity of the aggregates. Not determined.
the other hand, LLH-3-CP led to large and polydisperse
particles (B30 nm diameter), but with a unimodal distribution.
The large and polydisperse particles observed with the hydro-
genated derivatives suggest the formation of loose aggregates
rather than spherical micelles. Upon dilution, the size and the
shape of the particles of the hydrogenated LAH-4-AT and
LLH-3-CP do not significantly change, while at 1 mM, phase
separation into two particles was observed for LAF-4-AT,
similar to that observed for LAH-4-AT. The stronger self-
aggregation behaviour of the fluorinated nitroxides is in
agreement with their higher lipophilicity as demonstrated
by the partition coefficient values (Table 1). It has to be
emphasized that below 1 mM, no stable aggregates were
observed for any of the compounds.
six-membered nitroxides.34 The rationale for the more facile
oxidation of six-membered systems was that it was due to a
strong pyramidal distortion of the nitroxide moiety leading to
a larger spin density on the nitrogen atom.35 Finally, the redox
potentials for all the conjugated nitroxides were found to be
higher than the non-conjugated ones. Based on the DLS data
presented above, the formation of micellar aggregates at
200 mM is not likely to occur for the amphiphilic nitroxides,
suggesting that the observed increase in their redox potentials
is not due to self-aggregation. However, the steric hindrance
provided by the bulky carrier could cause shielding of the
nitroxide group, thereby limiting the interaction of the
aminoxyl group with the electrode surface and resulting in a
more difficult oxidation.
Cyclic voltammetry
Relative rates of reduction
Cyclic voltammetry (CV) was used to investigate the redox
behavior of the amphiphilic nitroxides (200 mM) in aqueous
solution. As expected, all the nitroxides underwent reversible
coupling corresponding to their direct oxidation to the
N-oxoammonium cation form.
The rates of reduction of the amphiphilic nitroxides using
ascorbate as reducing agent were measured and were normalized
with respect to 3-CP. For comparison, TEMPO, 4-AT and
4-AcT were also included, and the data are shown in Table 1.
As described in the literature, the rates of reduction were
found to be influenced by the ring size of the nitroxides, with
the five-membered ring being less reactive than the six-membered
ring nitroxides due to the conformational flexibility of the
latter compared to the former.36,37 Among the pyrrolidine
series, the parent compound is three times less reactive than
the amphiphilic derivatives, demonstrating that the anionic
carboxylate group decreases the reduction rate due to the
repulsive interaction between the negatively charged ascorbate
and 3-CP. Moreover, no significant difference was observed
between the LLF-3-CP and LLH-3-CP, suggesting that the
fluorinated chain has no effect on the reduction rate. Among
the piperidine series, TEMPO was found to be the less
reactive, with a reduction rate constant B23 times higher than
that of 3-CP, while we were unable to determine the reduction
rate of 4-AT due to its fast decomposition. The very high
reactivity of the amino compound, 4-AT, may be attributed to
the electrostatic attraction between the cationic nitroxide and
the anionic ascorbate, which supports the previous findings
by Morris et al.37 Acetylation of the amino group of 4-AT
(i.e., 4-AcT) significantly decreased the reactivity towards
ascorbate, with a relative constant of B107, which is B5 times
higher than the non-substituted TEMPO. This further
The peak separation (DEp) ranges from 58 to 66 mV,
approaching the theoretical Nernstian value (59 mV) and
indicating a kinetic reversibility of the oxidation process.
The redox potentials (E1/2) of the oxidation of the nitroxides
were estimated as half of the sum of the anodic (Epa) and
cathodic peak (Epc) potentials, and the data are shown in
Table 1. Among the six-membered ring nitroxides, 4-AT gave
the highest redox potential (E1/2 = 612 mV) while TEMPO
gave the lowest (E1/2 = 402 mV), indicating facile oxidation
for the latter. This is in agreement with previous studies
showing substituent effects on the redox potentials.33,34
Acetylation of the amino group of 4-AT (e.g., 4-AcT)
significantly decreased the redox potentials with a value of
E1/2 = 421 mV, further confirming the effect of the substituent
group on the redox potentials, but the presence of amide
bond does not significantly alter the redox properties of the
nitroxide compared to the non-substituted TEMPO. However,
the amphiphilic LAH-4-AT and LAF-4-AT exhibited higher
values than the related amido-derivative, 4-AcT. The higher
values for the lysinyl 3-CP derivatives LLH-3-CP and LLF-3-CP
compared to the 4-AT aspartic acid derivatives are in agreement
with the literature, confirming the ease of oxidation of the
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confirms that the electron-withdrawing inductive effect of a
substituent can increase the reactivity of the nitroxyl group.
Moreover, the amphiphilic LAH-4-AT and LAF-4-AT exhibited
similar values to their related amido derivative 4-AcT, indicating
that the acid aspartic-based carrier, regardless of the nature of
the chain, does not affect the reduction rate. However, a slight
increase in the reduction rate was observed for the lysinyl
derivatives compared to the 3-CP, and this is very likely due
to the suppression of repulsive electrostatic interactions. In
summary, we found that six-membered nitroxides are more
rapidly reduced than the five-membered nitroxides, consistent
with the literature,36,37 and that the nature of the substituent
on the nitroxide also affects the reduction rate through the
inductive effect and electrostatic interaction with the negatively
charged ascorbate. However, the ability of amphiphilic nitroxides
to self-aggregate does not seem to alter the reduction rate.
larger for 3-CP than for 4-AT, consistent with the faster
diffusion of the nitroxide with smaller size.
The effect of concentration on the EPR spectral profiles of
the conjugated nitroxides for both hydrogenated and fluorinated
carriers grafted to 3-CP and 4-AT were studied, and found to
show a different behaviour from that of the unconjugated
nitroxides. All spectra can be interpreted as a superimposed
signal of a narrow and a broad triplet corresponding to the
non-clustered and clustered radicals, respectively. Representative
EPR spectra for LLF-3-CP and LLH-3-CP at low and high
concentrations are shown in Fig. 3. Similar trends in spectral
profile at various concentrations were observed for LAH-4-AT
and LAF-4-AT (see Fig. S23 and S24, respectively) but the
triplet feature was still evident for LAF-4-AT. For the cluster
signal in the hydrogenated carrier, aN decreases by ca. 0.3 G,
while the linewidth increases by 1.3 G (see Table 3). The
calculated K1 from the 2-D simulation of the spectra (see
Experimental section for details) gave larger values for the
fluorinated compounds compared to the hydrogenated ones,
indicating a stronger tendency for aggregation for the former
compared to the latter. The larger linewidth and smaller aN for
Electron paramagnetic resonance spectroscopy (EPR) studies
EPR spectroscopy is commonly employed to study the
dynamics of complexation or aggregation of paramagnetic
species.38 The nitrogen hyperfine splitting constant (aN) is
affected by the polarity around the probe, the pyramidal
distortion of the nitroxide moiety as well as the formation of
hydrogen bonds with the solvent. An increase in the polarity
or hydrogen bonding favours the pseudo-ionic structure B,
thus resulting in a higher spin density on the nitrogen and
therefore larger aN values,39 while the linewidth broadening
results from hindered molecular motion due to microenvironment
viscosity, complexation and/or self-aggregation.
the fluorinated nitroxides indicate
a stronger magnetic
interaction between the nitroxides. This confirms the formation
of more compact and smaller aggregates, as already observed
by DLS measurements. Moreover, the observed singlet signal
for the clustered LLF-3-CP spectrum (Fig. 3) compared to
that of LAF-4-AT (Fig. S24) in which the triplet feature is
more evident, indicates that stronger magnetic interactions
between the nitroxides occurs within the aggregates of
LLF-3-CP. This demonstrates that the lysine carrier increases
the aggregation compared to the aspartic acid carrier based on
the calculated formation constants shown in Table 3. This is
consistent with the stronger self-aggregation properties of the
lysine derivatives compared to the aspartic acid ones, as
demonstrated by size particle measurements and partition
coefficients previously obtained in this work. As shown in
Fig. 4, the partition of nitroxides between their non-clustered
and clustered forms versus log[concentration] leads to a
sudden change in the slope of the graph for all the nitroxides
(see also Fig. S25 for plots as a function of concentration). At
this particular concentration, a change in the microenvironment
around the nitroxide occurs, which indicates the initial formation
of clusters. The change is observed at a concentration lower
Fig. S21 and S22 show the concentration dependence of the
aN and linewidths for the unconjugated nitroxides 4-AT and
3-CP. No superimposition of spectral lines was observed and
the linewidth varies continuously as a function of concentration.
The increase in linewidth and the lowering of aN are caused by
the encounters between radicals, as evidenced by the quadratic
concentration dependence. In both figures, the lines exhibit a
perfect parabolic fit. The curvature of the parabolic curve is
Fig. 3 X-band EPR spectra of the non-clustered and clustered LLF-3-CP (left) and LLH-3-CP (right) in PBS.
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Table 3 EPR parameters and formation constants for LAH-4-AT, LAF-4-AT, LLH-3-CP, and LLF-3-CP in PBS at 298 K
LAH-4-AT
Non-cluster
LAF-4-AT
Non-cluster
LLH-3-CP
Non-cluster
LLF-3-CP
Cluster
Cluster
Cluster
Non-cluster
Cluster
g
2.00551
17.06
0.43
2.00550
16.77
1.73
2.00549
17.07
0.44
2.00470
13.59
9.89
2.00528
16.07
0.23
2.00533
15.81
1.50
2.00530
16.08
0.25
2.00476
10.00
7.00
aN (G)
a (G)
K1 (L molꢁ1
)
—
247
—
1109
—
406
—
1642
not for 3-CP from 1 h to 24 h. Moreover, addition of 250 mM
of K3[Fe(CN)6] to convert the possibly formed hydroxyl-
amines back to the nitroxide only gave a small increase in
signal intensity for 4-AT and TEMPO but not for LAH-4-AT
(Fig. S27). This suggests that 4-AT, LAH-4-AT and TEMPO
may have been metabolized intracellularly and/or converted to
products other than hydroxylamines, while 3-CP remains
likely extracellular due to its anionic nature in solution. In
addition, cells lysed after 1 h incubation with 4-AT showed a
detectable EPR signal consistent with the permeability
of 4-AT compound (o10% relative intensity compared to
control, Fig. S27); however, no signal was detected in lysed
cells after 24 h, indicating further degradation of the nitroxide.
Fig. 4 Radical distributions as a function of logC showing the
varying degrees of aggregation (see also Fig. S25 for plots as a function
of concentration).
Cytotoxicity of nitroxides
As shown in Fig. 5 using BAEC, the cytotoxicity of the various
nitroxides was initially determined in the concentration range
of 75–1000 mM, and results show that the non-conjugated
nitroxides, TEMPO, 4-AT, 4-AcT, 3-CP and LLF-3-CP are
non-toxic, in agreement with the literature for the piperidine
nitroxides TEMPO, 4-AT and 4-AcT,41 while LAF-4AT,
LAH-4-AT and LLH-3-CP (up to 500 mM) were found to
induce moderate toxicity (i.e., >70% viability) at the
same concentration range. Similar toxicity behavior at high
concentrations was observed with an amphiphilic cyclic nitrone
comprised of the same fluorinated carrier, while the linear
nitrone derivative was found to be devoid of any toxicity.29
This confirms that conjugation of certain antioxidants with an
amphiphilic carrier may induce a little toxicity at high
than 1 mM for LLF-3-CP and LAF-4-AT while those of
LAH-4AT and LLH-3CP are B3.6 mM and B2.1 mM,
respectively, which could be correlated with the critical micellar
concentrations.
The micellisation of surfactants has been demonstrated by
EPR using nitroxides as probes, and it was shown that in the
premicellar concentration range, the aN values do not change
with the surfactant concentration while a decrease of aN is
observed below the cmc.39,40 In this case, as the nitroxide
moiety is covalently grafted onto the amphiphilic structure,
the EPR interpretation could be more complex and further
experiments need to be carried out to confirm whether the
change observed in the radical distribution actually corresponds
to a critical micellar concentration. The nature and the size of
the aggregates formed by this novel series of amphiphilic
nitroxides will be further investigated in the future by means
of neutron and X-ray scattering as well as analytical ultra-
centrifugation and surface tension measurements.
Stability of nitroxides with bovine aortic endothelial cells
(BAEC)
As shown in Fig. S26, the reduction of 4-AT, TEMPO and
LAH-4AT with ascorbic acid was fully reversed upon treatment
with K3[Fe(CN)6] while the very stable 3-CP was not reduced
in the presence of ascorbic acid, confirming the reduction rate
data presented above. The incubation of these four nitroxides
alone (25 mM) for 24 h in cell culture media but in the absence
of BAEC did not result in any significant decrease in their
EPR signal intensity, demonstrating their stability in the cell
culture media at 37 1C. However, incubation in the presence of
BAEC shows a significant decrease in signal intensity in the
extracellular media for 4-AT, LAH-4-AT and TEMPO but
Fig. 5 Cytotoxicity of various nitroxides. Cells were incubated in the
presence of nitroxides at concentrations 75, 300, 500 and 1000 mM for
24 h. Cell viability was measured using MTT assay (see experimental
for details). The y-axis corresponds to the % viability relative to
untreated cells. n = 3–5.
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concentration. In spite of the higher toxicity observed in vitro for
the pyrrolidine nitroxides compared to the piperidine
nitroxides,12 3-CP was found to be non-toxic within the concen-
tration range used in this study. The acute toxicity of LLH-3-CP
observed at 1000 mM is most likely due to its detergent property,
which could result to cell membrane lysis. Indeed, no toxicity was
observed with its fluorinated analog at the same concentration.
preventing hydrogen peroxide-induced cytotoxicity. Moreover,
previous studies41,42 showed that while 4-AT was found to be
rapidly reduced to its hydroxylamine form and remains inside
the cell in this reduced form, 4-AcT was found to be slowly
reduced, and the concentration of its hydroxylamine form in
the cell is lower. The absence of antioxidant property of
TEMPO was explained by its reduction to its amine form
and the weak antioxidant properties of secondary amines.8
The following trend, in order of decreasing protection of
BAEC exposed to lethal concentration of hydrogen peroxide,
was observed: 4-AT > 4-AcT > TEMPO, and is similar to
those previous observed.
Cytoprotective properties
The effect of amphiphilic nitroxides on H2O2-treated BAEC
was investigated in the concentration range of 75–500 mM and
compared to that of the non-conjugated derivatives. We also
used the parent compounds 4-AT and 3-CP as controls in
order to know whether the amphiphilic-mediated targeting
strategy is relevant when used with nitroxides. These parent
compounds, however, are expected to be charged in buffer
solution that could affect their membrane permeability
compared to the neutral conjugated analogues, so we therefore
also used the uncharged 4-AcT and TEMPO to discount the
charge effects.
Hydrogen peroxide diffuses through cell membranes, and at
unregulated concentrations may induce cellular injury, leading
to apoptosis or necrosis.43,44 The mechanism of antioxidant
action of nitroxides (or its reduced hydroxylamine form)
against H2O2-induced toxicity is not well understood but is
believed to be due to its direct reaction with HOꢀ (via Fenton
reaction). The reported rate constants for these reactions are in
the order of 109 Mꢁ1 sꢁ1 45
and the observations by Czespas
,
et al.41 are in agreement with this hypothesis. Moreover, the
ability of nitroxides to oxidize low-valent transition metal ions
Cu(I) and Fe(II) could also limit the production of HOꢀ via a
Fenton-type reaction. Also, the fact that cell-permeable
In general, the 5-membered ring nitroxides showed no
protective effects in the concentration range tested, unlike
the 6-membered ring nitroxides (Fig. 6). In addition, varying
degrees of protection were observed among the 6-membered
ring nitroxides tested, and 4-AT which bears an amino sub-
stituent at the C-4 position, was found to be the most
protective, followed by the C-4 acetamide substituted com-
pound (4-AcT), and by LAF-4-AT. However, the compounds
LAH-4-AT and TEMPO exhibited the lowest protection
among the 6-membered ring nitroxides tested. Czepas et al.41
showed that substitution at the C-4 position of piperidine
nitroxide is an important parameter affecting its biological
property. For example, 4-AcT and 4-AT are more potent than
TEMPO and its hydroxylated derivative, TEMPOL, in
13,46
nitroxides can prevent DNA damage induced by H2O2
and that nitroxides and hydroxylamines equally prevented
DNA damage45 suggest that the protection offered by nitroxides
or its metabolic product (i.e., hydroxylamine) are mostly
intracellular in nature.46 Moreover, nitroxides in fact exhibit
anti-apoptotic activity,31 perhaps through its SOD mimetic
activity, oxidation of semiquinone radicals, and modulation of
NO levels via sequestration of superoxide radical anion. It can
therefore be assumed that the antioxidant activity of nitroxides
encompasses complex mechanisms that can involve direct or
indirect radical reaction with exogenously and endogenously
generated radicals during the 24 h of cell treatment with H2O2.
As we demonstrated above, the rate of reduction of 4-AT by
ascorbate was much faster than those of the other piperidine
nitroxides; such an observation could explain the high efficacy
of 4-AT against cytotoxicity. In contrast, the pyrrolidine
compounds were found to be slowly reduced by ascorbate
and exhibited no protective activity. The higher potency of
4-AT compared to its amido derivative 4-AcT indicates that
the nature of the substituent plays a major role in cytoprotection.
The fact that 4-AcT and LAF-4-AT did not show any
significant difference in their protective properties suggests
that the amphiphilic group does not play a major role in
cytoprotection and that their lipophilicity does not affect their
biological activity. This result is quite surprising since we
demonstrated that grafting the nitrones PBN and DMPO onto
the same fluorinated carrier significantly increases the potency
of the parent nitrones in preventing oxidant-induced toxicities.23,29
Also worth noting is that in our procedure, cells were challenged
with H2O2 24 h after being treated with the nitroxides. This
suggests that the protection afforded by the nitroxides could
be mostly provided by hydroxylamine17 (or its metabolic
products, where such a scenario is possible during 24 h), and
the fact that nitroxides cannot be restored from the cytosolic
fractions after 24 h of incubation.
Fig. 6 Cytoprotective properties of various nitroxides (0, 75, 300 and
500 mM) against hydrogen peroxide induced cell death. Cells were
incubated in the absence or presence of nitroxides for 24 h before being
exposed to 300 mM hydrogen peroxide for another 24 h. Cell viability
was measured using MTT assay (see Experimental section for details).
The y-axis corresponds to the % viability relative to untreated cells. *
Significantly different from 300 mM hydrogen peroxide treatment by
t-test, p o 0.05, n = 2–4.
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(C), 67.1 (CH2), 51.1, 47.2 (CH), 39.7, 31.8, 29.5, 29.2 (CH2),
28.1 (CH3), 26.8, 22.6 (CH2), 14.1 (CH3).
Conclusions
A novel series of amphiphilic nitroxides possessing a lactobionic
polar head, a hydrophobic tail, and an amino acid as scaffold
conjugated to 4-amino TEMPO or 3-carboxyproxyl were
synthesized. Fluorinated amphiphilic nitroxides LLF-3-CP
and LAF-4-AT led to small and monodisperse aggregates of
B5–6 nm diameter while the hydrogenated 3-CP derivative led
to larger micellar aggregates of B30 nm diameter, and its
4-AT derivative led to polydisperse aggregates. The stronger
self-aggregation properties of the fluorinated derivatives
compared to hydrogenated ones were confirmed by EPR
experiments. The large linewidth and small aN for the fluorinated
nitroxides suggests a more compact cluster where the magnetic
interaction between the nitroxides are rather strong. EPR
results also show that the lysine carrier increases the clustering
compared to the aspartic acid.
N-(2,3,4,6,20,30,40,60-O-Acetyl-lactobionyl)-b-(tert-butyloxy-
carbonyl)-L-aspartyl-octylamide (2). Compound
1 (0.8 g,
1.53 ꢄ 10ꢁ3 mol) was dissolved in a DEA–CH3CN 1 : 9 (v/v)
mixture at room temperature. After 1 h of being stirred, the
solvent was evaporated under vacuum to give the corresponding
amino derivative as a yellow oil (0.445 g, 1.49 ꢄ 10ꢁ3 mol,
97%). Lactobionolactone (0.713 g, 1.99 ꢄ 10ꢁ3 mol), prepared
according to a published procedure, and the resulting amino
compound were dissolved in 2-methoxyethanol with TEA (pH
8–9). The mixture was stirred at 65 1C under argon for 28 h
until complete consumption of the amino derivative. Then, the
solvent was evaporated under vacuum and the residue was
added to a solution of Ac2O–pyridine 1 : 1 (v/v) at 0 1C. After
12 h of being stirred, the mixture was poured into cold 1 N
HCl and extracted with CH2Cl2 (3ꢄ). The organic layer was
washed with brine, dried over Na2SO4, and concentrated
under vacuum. The crude mixture was purified by flash
chromatography (EtOAc–cyclohexane 4 : 6 v/v) to give
compound 2 (0.585 g, 5.99 ꢄ 10ꢁ4 mol, 40%) as a white foam.
Rf 0.40 (EtOAc–cyclohexane 6 : 4 v/v). mp 48.2–49.3 1C
(decomposition). [a]2D5 +52.0 (c l in CH2Cl2). dH(250 MHz,
CDCl3) 7.52 (1H, d, J = 8.3 Hz), 6.6 (1H, t, J = 5.6 Hz), 5.49
(1H, m), 5.38 (2H, m), 5.11 (2H, m), 4.99 (1H, m), 4.66–4.52
(3H, m), 4.15–3.98 (5H, m), 3.18 (2H, m), 2.93 (1H, dd, J =
3.6, 17.0 Hz), 2.42 (1H, dd, J = 6.4, 17 Hz), 2.23–1.98 (24H,
m), 1.43 (9H, s), 1.25 (12H, s), 0.86 (3H, t, J = 6.5 Hz).
dC(62.86 MHz, CDCl3) 172.1, 170.5, 170.2, 170.1, 170.1, 169.7,
169.6, 169.5, 169.2, 168.0 (CO), 101.5 (CH), 81.9 (C), 78.0,
72.3, 71.1, 71.0, 69.8, 69.6, 68.9, 66.9 (CH), 61.4, 61.1 (CH2),
49.0 (CH), 39.9, 31.8, 29.3, 29.2 (CH2), 28.0 (CH3), 26.8, 22.6,
(CH2) 20.8, 20.7, 20.6, 20.5, 14.1 (CH3). MS (ESI+, m/z)
977.3 [(M + H)+], 994.3 [(M + NH4)+], 999.3 [(M + Na)+],
1015.3 [(M + K+)].
Cyclic voltammetry showed that all the nitroxides underwent
reversible couple corresponding to their direct oxidation to the
N-oxoammonium cation form. As previously reported, we
observed the ease of oxidation of the six-membered ring
nitroxides and that the oxidation potentials being increased
upon conjugation to an amphiphilic carrier. The relative rates
of reduction of the nitroxides by ascorbate showed that
piperidine nitroxides are more rapidly reduced than pyrrolidine,
and that the nature of the substituent on the nitroxide also
affects the reduction rate through inductive and electrostatic
effects. However, no correlation between the amphiphilicity of
the nitroxide and the reduction rate was observed. While the
six-membered ring nitroxides exhibited cytoprotective activity
against H2O2-induced cell death, the five-membered ring
nitroxides showed no protective effect. The order of decreasing
cytoprotective ability for the piperidine series is: 4-AT >
LAF-4-AT = 4AcT > LAH-4-AT > TEMPO. This suggests
that the amphiphilic group does not play a major role in
cytoprotection of nitroxide compounds, contrary to what was
observed for the nitrones PBN and DMPO, in which grafting
onto the fluorinated amphiphilic carrier led to increased
cytoprotection by the parent nitrone.
N-(2,3,4,6,20,30,40,60-O-Acetyl-lactobionyl)-b-(4-amido-TEMPO)-
L-aspartyl-octylamide (3). At 0 1C, compound 2 (0.500 g,
5.12 ꢄ 10ꢁ4 mol) was dissolved in a TFA–CH2Cl2 4 : 6
(v/v) mixture. After 2 h of being stirred, the solution was
concentrated under vacuum to give the corresponding acid
derivative as a white powder (0.434 g, 4.71 ꢄ 10ꢁ4 mol, 92%).
The resulting acid derivative (0.3 g, 3.26 ꢄ 10ꢁ4 mol), EDC
(0.075 g, 3.91 ꢄ 10ꢁ4 mol), 4-AT (0.024 g, 4.24 ꢄ 10ꢁ4 mol)
and a catalytic amount of HOBt were dissolved in dry CH2Cl2
with DIEA (pH = 8–9) under argon. The mixture was stirred
for 36 h at room temperature and then the solvent was
evaporated under vacuum. The crude mixture was purified by
flash chromatography (EtOAc–cyclohexane 7 : 3 v/v) followed by
size-exclusion chromatography (CH2Cl2–MeOH 1 : 1 v/v) to
give compound 3 (0.174 g, 1.62 ꢄ 10ꢁ4 mol, 50%) as an orange
powder. Rf 0.42 (EtOAc). mp 73.1–74.2 1C (decomposition).
Experimental section
Syntheses
N-(9-Fluorenylmethyloxycarbonyl)-b-(tert-butyloxycarbonyl)-
L-aspartyl-octylamide (1). A mixture of octylamine (0.753 g,
5.83 ꢄ 10ꢁ3 mol), FmocAsp(OtBu)OH (2 g, 4.86 ꢄ 10ꢁ3 mol),
DCC (1.2 g, 5.83 ꢄ 10ꢁ3 mol), and HOBt in dry CH2Cl2 was
stirred for 2 h at room temperature and then concentrated
under vacuum. The crude mixture was purified by flash chromato-
graphy (EtOAc–cyclohexane 2 : 8 v/v) to give compound 1
(2.34 g, 4.47 ꢄ 10ꢁ3 mol, 92%) as a white powder. Rf 0.53
(EtOAc–cyclohexane 4 : 6 v/v). mp 117 1C. [a]2D5 +13.7 (c l in
CH2Cl2). dH(250 MHz, CDCl3) 7.80 (2H, d, J = 7.6 Hz), 7.61
(2H, d, J = 7.3 Hz), 7.47–7.29 (4H, m), 6.48 (1H, m), 5.98 (1H,
d, J = 7.7 Hz), 4.47 (3H, m), 4.25 (1H, t, J = 6.8 Hz), 3.26 (2H,
m), 2.98–2.53 (2H, m), 1.48 (12H, s), 1.29 (9H, s), 0.9 (3H, t,
J = 7.1 Hz). dC(62.86 MHz, CDCl3) 171.3, 170.2, 155.4 (CO),
143.7, 141.3 (C), 127.8, 127.1, 125.0, 120.1, 120.0 (CH), 82.9
l
max(CH2Cl2)/nm 229. MS (ESI+, m/z) 1074.4 [(M + H)+],
1096.4 [(M + Na)+], 1112.3 [(M + K)+].
N-Lactobionyl-b-(4-amido-TEMPO)-L-aspartyl-octylamide (4).
Compound 3 (0.160 g, 1.49 ꢄ 10ꢁ4 mol) was dissolved under
argon in MeOH and a catalytic amount of sodium methoxide
was added. The mixture was stirred for 4 h and HCl (1 N) was
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added dropwise to neutralize the solution. The solvent was
evaporated under vacuum and the crude mixture was purified
by size-exclusion chromatography (MeOH) to give compound
4 (0.084 g, 1.12 ꢄ 10ꢁ4 mol, 90%) as an orange powder. Rf 0.5
(EtOAc–MeOH–H2O 7 : 2 : 1 v/v/v). mp 88.6–89.4 1C
(decomposition). [a]2D5 +26.3 (c l in MeOH). lmax(MeOH)/
nm 229. HR-MS (ESI+, m/z) calcd for C33H62N4O14
[(M + H)+]: 738.4257, found 738.4266.
N-(2,3,4,6,20,30,40,60-O-Acetyl-lactobionyl)-Ne-(carboxyproxyl)-
L-lysinyl-octylamide (7). At 0 1C, compound 6 (0.3 g, 2.81 ꢄ
10ꢁ4 mol) was dissolved in ethanol–acetic acid 99 : 1 (v/v) and
0.018 g of 10% Pd/C was added portionwise with stirring. The
reaction mixture was exposed to a hydrogen atmosphere for
12 h (8 bars). After filtration of the catalyst through a pad of
Celite, the solvent was evaporated under vacuum to give the
corresponding amino derivative (0.256 g, 2.75 ꢄ 10ꢁ4 mol).
The resulting amino (0.240 g, 2.57 ꢄ 10ꢁ4 mol), EDC (0.049 g,
2.57 ꢄ 10ꢁ3 mol), 3-CP (0.040 g, 2.15 ꢄ 10ꢁ3 mol) and a
catalytic amount of HOBt were dissolved in dry CH2Cl2 with
DIEA (pH = 8–9) under argon. The mixture was stirred for
18 h at room temperature and the solvent was evaporated
under vacuum. The crude mixture was purified by flash
chromatography (EtOAc) and by size-exclusion chromatography
(CH2Cl2–MeOH 1 : 1 v/v) to give compound 7 (0.076 g,
0.69 ꢄ 10ꢁ4 mol, 32%) as a yellow foam. Rf 0.33 (EtOAc).
mp 98.5–99.4 1C (decomposition). [a]2D5 +10.5 (c l in CH2Cl2).
N-(tert-Butyloxycarbonyl)-Ne-(benzyloxycarbonyl)-L-lysinyl-
octylamide (5). The synthetic procedure was essentially the
same as for compound 1. A mixture of octylamine (1.22 g,
9.46 ꢄ 10ꢁ3 mol), BocLys(Z)OH (3 g, 7.88 ꢄ 10ꢁ3 mol), DCC
(1.95 g, 9.46 ꢄ 10ꢁ3 mol) and a catalytic amount of HOBt in
dry CH2Cl2 was stirred for 24 h at room temperature and then
concentrated under vacuum. The crude mixture was purified
by flash chromatography (EtOAc–cyclohexane 4 : 6 v/v) to
give compound 5 (2.88 g, 5.86 ꢄ 10ꢁ3 mol, 74%) as a white
powder. Rf 0.46 (EtOAc–cyclohexane 5 : 5 v/v). mp 78 1C. [a]D25
ꢁ6.0 (c l in CH2Cl2). dH(250 MHz, CDCl3) 7.34 (5H, s), 6.49
(1H, m), 5.36 (1H, d, J = 7.87 Hz), 5.09 (3H, s), 4.05 (1H, d,
J = 5.91 Hz), 3.19 (4H, m), 1.86–1.11 (6H, m), 1.43 (9H, s),
1.27 (12H, s), 0.88 (3H, t, J = 6.52 Hz). dC(62.86 MHz,
CDCl3) 172.4, 156.6 (CO), 136.6 (C), 128.5, 128.1 (CH), 80.4
(C), 66.7 (CH2), 54.4 (CH), 31.9, 31.8, 29.5, 29.3, 29.2 (CH2),
28.2 (CH3), 26.9, 25.6, 25.0, 22.7, 22.6 (CH2), 14.1 (CH3).
l
1119.7 [(M
[(M + K)+].
max(CH2Cl2)/nm 231. MS (ESI+, m/z) 1102.7 [(M + H)+],
+ +
NH4)+], 1124.6 [(M Na)+], 1140.7
N-Lactobionyl-Ne-(carboxyproxyl)-L-lysinyl-octylamide (8).
Compound 7 (0.060 g, 5.45 ꢄ 10ꢁ5 mol) was dissolved under
argon in MeOH and a catalytic amount of sodium methoxide
was added. The mixture was stirred for 4 h and HCl (1 N) was
added dropwise to neutralize the solution. The solvent was
evaporated under vacuum and the crude mixture was purified
by size-exclusion chromatography (MeOH), to give the
compound 8 (0.040 g, 5.23 ꢄ 10ꢁ5 mol, 96%) as a yellow
powder. Rf 0.41 (EtOAc–MeOH–H2O 7 : 2 : 1 v/v/v). mp
118.2–119.4 1C (decomposition). [a]2D5 +19.0 (c 0.l in MeOH).
N-(2,3,4,6,20,30,40,60-O-Acetyl-lactobionyl)-Ne-(benzyloxy-
carbonyl)-L-lysinyl-octylamide (6). At 0 1C, compound 5
(2.86 g, 5.82 ꢄ 10ꢁ3 mol) was dissolved in a TFA–CH2Cl2
3 : 7 (v/v) mixture. After 6 h of being stirred, the solvent was
evaporated under vacuum to give the corresponding amino
derivative (2.13 g, 5.47 ꢄ 10ꢁ3 mol). The resulting amino
derivative (2 g, 5.13 ꢄ 10ꢁ3 mol) and lactobionolactone
(1.90 g, 5.29 ꢄ 10ꢁ3 mol) were dissolved in 2-methoxyethanol
with TEA, and the mixture was stirred at 65 1C under argon
for 24 h. Then, the solvent was evaporated under vacuum and
the residue was added to a solution of Ac2O–pyridine 1 : 1
(v/v) at 0 1C. After 12 h of being stirred, the mixture was
poured into cold 1 N HCl and extracted with CH2Cl2 (3ꢄ).
The organic layer was washed with brine, dried over Na2SO4,
and concentrated under vacuum. The crude mixture was
purified by flash chromatography (EtOAc–cyclohexane 4 : 6 v/v)
to give compound 6 (2.8 g, 2.62 ꢄ 10ꢁ3 mol, 50%) as a white
foam. Rf 0.20 (EtOAc–cyclohexane 6/4: v/v). mp 62 1C
(decomposition). [a]2D5 +5.4 (c l in CH2Cl2). dH(250 MHz,
CDCl3) 7.36 (5H, s), 6.61 (1H, d, J = 8.0 Hz), 6.08 (1H, m),
5.54 (1H, m), 5.38 (2H, m), 5.16–5.10 (4H, m), 5.01 (1H, dd,
J = 3.4, 10.0 Hz), 4.61 (1H, d, J = 7.8 Hz), 4.54 (1H, dd,
J = 2.9 Hz, 12.5 Hz), 4.18 (4H, m), 3.91 (1H, t, J = 6.6 Hz),
3.20 (4H, m), 2.24–2.00 (24H, m), 1.90–1.38 (6H, m), 1.28
(12H, s), 0.89 (3H, t, J = 6.5 Hz). dC(62.86 MHz, CDCl3)
170.6, 170.5, 170.2, 170.1, 170.0, 169.8, 169.3, 169.2, 167.5,
156.6 (CO), 136.7 (C), 128.5, 128.1, 128.0, 101.7, 77.9, 72.6,
71.1, 70.9, 69.9, 69.3, 68.9, 66.8 (CH), 66.5, 61.2, 61.0 (CH2),
52.7 (CH), 39.8, 31.8, 29.4, 29.2, 26.9, 22.6, 22.2 (CH2), 20.9,
20.8, 20.7, 20.6 20.5, 14.1 (CH3). MS (ESI+, m/z) 1068.4 [(M
+ H)+], 1085.4 [(M + NH4)+], 1090.4 [(M + Na)+], 1106.4
[(M + K)+].
l
max(MeOH)/nm 231. MS (ESI+, m/z) 766 [(M + H)+], 783
[(M + NH4)+], 788 [(M + Na)+], 804 [(M + K)+].
Determination of logk0W values
Compounds were dissolved in MeOH at 1.0 mg mLꢁ1 and
were injected onto a Microsorb C18 column (250 mm ꢄ
4.6 mm, 5 mm). The compounds were eluted at various MeOH
and water ratios using a flow rate of 0.8 mL minꢁ1. The
column temperature was 25 1C, and the UV detector wave-
length was l = 230 nm. Linear regression analyses were
performed on 3 data points (from 8 : 2 to 6 : 4 v/v) for LAH-
4-AT, LAF-4-AT, LLH-3-CP and LLF-3-CP (r2 > 0.9992);
four points (from 8 : 2 to 4 : 6 v/v) for 4-AT and 3-CP
(r2 > 0.9979). logk0 values were calculated by using the
equation: logk0 = log((t ꢁ t0)/t0), where t is the retention time
of the nitroxide and t0 is the elution time of MeOH, which is
not retained on the column.
Particle size analyses
The hydrodynamic particle size distributions and polydispersity
of amphiphilic nitroxides at different concentrations were
determined by using a Zetasizer Nano-S model 1600 (Malvern
Instruments Ltd., UK) equipped with a He–Ne laser (l = 633 nm,
4.0 mW). In a typical experiment, stock solutions (10 mM) in
milli-Q water (resistivity 18.2 mO cm) were prepared and
stored at room temperature overnight before the measurements.
On the day of the experiment, solutions were passed through a
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0.45 mm filter, a low-volume quartz batch volume was filled
with 100 mL of the stock solution, and the size of the particles
was measured 1 h after filtration, and then solutions were
gradually diluted. The time-dependent correlation function of
the scattered light intensity was measured at a scattering angle
of 1731 relative to the laser source. The hydrodynamic radius
(R) of the particles was estimated from their diffusion coefficient
(D) using the Stokes–Einstein equation, D = kBT/6pZR,
where kB is the Boltzmann constant, T is the absolute
temperature, and Z is the viscosity of the solvent.
stability constants for different species even if the N values
are not the same. The 2-D simulation program decomposes
simultaneously a set of superimposed spectra recorded at
different concentration of the radicals. In this procedure
10 EPR parameters (e.g., g-factor, aN, a, b, g relaxation) of
the two species (i.e., non-cluster and cluster) and the KN
formation constant were adjusted using a simulation program
for a set of spectra including 11 spectra with the concentration
0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 25 mM,
respectively. The value of N was varied between 2 and 80 to
test which equilibrium gives the best fit. The optimum was
found in the range 40–80, but the regression factor showed
only a slight variation for large N values.
Cyclic voltammetry
Cyclic voltammetry was performed on a potentiostat and
computer-controlled electroanalytical system. Electro-chemical
measurements were carried out in a 10 mL cell equipped with
a glassy carbon working electrode, a platinum-wire auxiliary
electrode, and a Ag/AgCl reference electrode. Solutions were
degassed by bubbling with nitrogen gas before recording the
voltamograms at a scan rate (n) of 0.1 V sꢁ1. The nitroxide
concentration was 200 mM in double-distilled water and the
supporting electrolyte was 0.15 M NaCl.
Cell viability and stability studies
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT)
reduction assay. Cytotoxicity of H2O2 and cytoprotective
properties of the nitroxides was assessed using MTT assay.
In a typical experiment, BAEC cells were incubated with
nitroxides for 24 h in DMEM supplemented with 0.5% FBS
in 24-well culture plates at 37 1C. After incubation, a
known concentration of H2O2 was added to the well
plates and incubated for an additional 24 h. A 0.5 mL MTT
solution (0.45 mg mLꢁ1 MTT in DMEM supplemented
with 0.5% FBS) was then added to each well. Cells were
incubated for another 2 h at 37 1C. After the 2 h incubation,
the media were removed and wells were rinsed once with
DPBS. A 0.3 mL mixture of dimethyl sulfoxide, isopropanol
and deionized water (1 : 4 : 5) was added to each well at
room temperature to solubilize the formazan crystals. The
dissolved formazan was then transferred into semi-micro
cuvettes, and the absorbance was measured at 570 nm using a
spectrophotometer.
EPR spectroscopy
EPR spectra were recorded at room temperature using an
X-band spectrometer with high sensitivity resonator. General
instrument settings for kinetic experiments were as follows:
microwave power, 10 mW; microwave frequency, 9.87 GHz;
modulation frequency, 100 kHz; modulation amplitude, 1.0 G;
sweep width, 120 G; time constant, 10.240 ms; sweep time,
20.40 s. General instrument settings for concentration
dependence experiments were as follows: microwave power,
1 mW; microwave frequency, 9.87 GHz; modulation frequency,
100 kHz; modulation amplitude, 1 G; time constant, 20.5 ms;
scan time, 41 s. Measurements were performed using 50 mL
capillary tubes.
Stability studies. In six-well plates, BAEC cells were
incubated with 25 mM of the nitroxide for a period of 1 and
24 h. Cells were collected and lysed by sonication. A portion of
the cell culture media and lysates were then transferred to
50 mL capillary tubes and the EPR spectra were recorded at
room temperature. A solution of 250 mM K3[Fe(CN)6] was
then added to the remaining media and lysates to restore any
reduced compound that was formed, and the EPR spectra was
taken again. Signal intensities were obtained for each experiment
and were compared to the signal intensity of 25 mM of the
respective nitroxides incubated alone for 24 h in the absence
of BAEC.
All kinetic experiments were performed in PBS buffer
solution. In a typical experiment, to a solution of nitroxide
(0.5 mM), ascorbate (5 mM) was added and kinetic measurement
was initiated 40 s after the addition of ascorbate. The decrease
in the area of the lowest field peak was monitored by EPR as a
function of time over a period of 100–900 s. All the data were
the average of two measurements. The rates of reduction
were calculated using pseudo-first-order rate law. For the
concentration dependence experiments, the nitroxides were
dissolved in PBS buffer, sonicated for 5 min, and left to stand
for 1 h before measurement.
The concentration effect can be well described by an
equilibrium between the non-clustered (R) and the clustered
radicals (RN).47 These two species were applied in the
2D- simulation of the spectra according to the following
equation: N ꢄ R 2 RN, with the formation constant
expressed as KN = [RN]/RN, where N is the number of
nitroxides in the cluster. Due to the exponential dependence
of KN with N, K1 was calculated instead as defined by the
relation: K1Nꢁ1 = KN where N ꢁ 1 corresponds to the number
of steps to form the cluster from the individual nitroxides.
When logK is divided by N ꢁ 1, one can obtain the logarithm
of the individual step. In this case, one can compare the
Statistical analysis. Statistical analysis was performed
using Student’s t-test. Statistical significance was considered
at P o 0.05.
Acknowledgements
This work is supported by NIH grant HL081248. The Ohio
Supercomputer Center (OSC) is acknowledged for generous
computational support of this research. FC is the recipient of a
´
fellowship from the ‘‘Region Provence Alpes Cote d’Azur’’
and Targeting System Pharma.
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1920 | New J. Chem., 2010, 34, 1909–1920