EPR study of dialkyl nitroxides as probes to investigate the exchange of solutes between the ligand shelf of monolayers of protected gold nanoparticles and aqueous solutions

Article abstract of DOI:10.1021/ja048554f

EPR spectroscopy has been used to study the interaction of para-substituted benzyl hydroxyalkyl nitroxides with the monolayer of water-soluble protected gold cluster made by a short alkyl chain and a triethylene glycol monomethyl ether unit. The inclusion

Full text of DOI:10.1021/ja048554f

Published on Web 07/08/2004
EPR Study of Dialkyl Nitroxides as Probes to Investigate the
Exchange of Solutes between the Ligand Shell of Monolayers
of Protected Gold Nanoparticles and Aqueous Solutions
Marco Lucarini,*,† Paola Franchi,† Gian Franco Pedulli,† Paolo Pengo,‡
Paolo Scrimin,‡ and Lucia Pasquato*,§
Contribution from the Department of Organic Chemistry “A. Mangini”, UniVersity of Bologna,
Via San Donato 15, 40127, Bologna, Italy; Department of Chemical Sciences and ITM-CNR,
PadoVa Section, UniVersity of PadoVa, Via Marzolo 1, 35131 PadoVa, Italy; and
Department of Chemical Sciences UniVersity of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy
Received March 12, 2004; E-mail: lucarini@alma.unibo.it; pasquato@dsch.univ.trieste.it
Abstract: EPR spectroscopy has been used to study the interaction of para-substituted benzyl hydroxyalkyl
nitroxides with the monolayer of water-soluble protected gold cluster made by a short alkyl chain and a
triethylene glycol monomethyl ether unit. The inclusion of nitroxide probes in the more hydrophobic
environment of the monolayer gave rise to a reduction of the value of both nitrogen and â-proton hyperfine
splittings. The spectra also showed selective line broadening attributed to modulation of the spectroscopic
parameters as the result of exchange between free and complexed nitroxide. The rate constants were
obtained by analyzing the EPR line shape variations as functions of nanoparticle concentration and
temperature. This represents, to the best of our knowledge, the first determination of rate constants for the
solubilization of organic substrates in a monolayer-protected cluster.
systems6 has expanded their field of application also to this
solvent where hydrophobic interactions are dominant.
Introduction
Hydrophobic interactions dominate many important processes
such as aggregation of surfactants and partition of biomolecules
in biological membranes.1 Interest in membrane mimetic systems
stems from their ability to provide relevant features pertinent
to natural membranes but also from the possibility to introduce
unnatural functional groups thus expanding the range of their
applications.2
Monolayer-protected nanoparticles (like gold nanoparticles,
Au-MPC) constitute one additional example of membrane
mimetic systems.3 They present the peculiarity of an extremely
slow exchange of the monomers and of a limited mobility in
the monolayer, thus allowing us to define precisely radial
subregions of different polarity.3,4 This limited mobility is at
the basis of their behavior as multivalent systems showing
cooperativity in the recognition of substrates5a or in performing
catalytic processes.5b The recent accessibility of water-soluble
EPR spectroscopy has been recently utilized to obtain useful
information on the interaction of nitroxides with gold nano-
particles7 and to investigate the mechanism of the place-
exchange reaction of thiols on the covering monolayer.8,9
In recent studies on complexation of radical species in
cyclodextrins,10a-c calixarenes,10d and micelles10e in aqueous
solutions, we have found benzyl tert-butyl nitroxide and related
dialkyl nitroxides to be very suitable probes to investigate host-
guest interactions. Evidence for the formation of paramagnetic
complexes between these radicals and the host systems was
provided by large spectral changes (a(N) and a(2Hâ)) due to
the less polar environment experienced by the radical guest and
to conformational changes occurring upon complexation. The
EPR spectra also showed a strong line width dependence on
(6) (a) Kanaras, A. G.; Kamounah, F. S.; Schaumburg, K.; Kiely, C. J.; Brust,
M. Chem. Commun. 2002, 2294-2295 and references therein. (b) Zheng,
M.; Davidson, F.; Huang, X. J. Am. Chem. Soc. 2003, 125, 7790-7791.
(c) Pengo, P.; Polizzi, S.; Battagliarin, M.; Pasquato, L.; Scrimin, P. J.
Mater. Chem. 2003, 13, 2471-2478.
(7) (a) Templeton, A. C.; Hoestler, M. J.; Warmoth, E. K.; Chen, S.; Hartshorn,
C. M.; Krishnamurthy, V. M.; Forbes, M. D. E.; Murray, R. W. J. Am.
Chem. Soc. 1998, 120, 4845-4849. (b) Zhang, Z.; Berg, A.; Levanon, H.;
Fessenden, R. W.; Meisel, D. J. Am. Chem. Soc. 2003, 125, 7959-7963.
(8) Chechik, V.; Wellsted, H. J.; Korte, A.; Gilbert, B. C.; Caldararu, H.; Ionita,
P.; Caragheorgheopol, A. Faraday Discuss. 2004, 125, 279-291.
(9) Ionita, P.; Caragheorgheopol, A.; Gilbert, B. C.; Chechik, V. J. Am. Chem.
Soc. 2002, 124, 9048-9049.
(10) (a) M. Lucarini, M.; Luppi, B.; Pedulli, G. F.; Roberts, B. P. Chem.sEur.
J. 1999, 5, 2048-2054. (b) Franchi, P.; Lucarini, M.; Pedulli, G. F. Angew.
Chem., Int. Ed. 2003, 42, 1842-1845. (c) Franchi, P.; Lucarini, M.;
Mezzina, E.; Pedulli, G. F J. Am. Chem. Soc. 2004, 126, 4343-4354. (d)
Franchi, P.; Lucarini, M.; Pedulli, G. F.; Sciotto, D. Angew. Chem., Int.
Ed. 2000, 39, 263-266. (e) Brigati, G.; Franchi, P.; Lucarini, M.; Pedulli,
G. F.; Valgimigli, L. Res. Chem. Intermed. 2002, 28, 131-141.
† University of Bologna.
‡ University of Padova.
§ University of Trieste.
(1) (a) Ben-Naim, A. In Hydrophobic Interactions; Plenum Press: New York,
1980. (b) Tanford, C. In The Hydrophobic Effect, 2nd ed.; Wiley: New
York, 1980.
(2) (a) Hoffmann, H.; Ebert, G. Angew. Chem., Int. Ed. Engl. 1988, 27, 902-
912. (b) Fuhrhop, J.-H.; Koening, J. Membranes and Molecular Assemblies,
The Synkinetic Approach; Royal Society of Chemistry: Cambridge, 1994.
(3) Badia, A.; Lennox, B. R.; Reven, L. Acc. Chem. Res. 2000, 33, 475-481.
(4) (a) Hostetler, M. J.; Green, S. J.; Stokes, J. J.; Murray, R. W. J. Am. Chem.
Soc. 1996, 118, 4212-4213. (b) Ingram, R. S.; Hostetler, M. J.; Murray,
R. W. J. Am. Chem. Soc. 1997, 119, 9175-9178. (c) Hasan, M.; Bethell,
D.; Brust, M. J. Am. Chem. Soc. 2002, 124, 1132-1133.
(5) (a) Fantuzzi, G.; Pengo, P.; Gomila, R.; Hunter, C. A.; Pasquato, L.; Scrimin,
P. Chem. Commun. 2003, 1004-1005. (b) Pasquato, L.; Rancan, F.;
Scrimin, P.; Mancin, F.; Frigeri, C. Chem. Commun. 2000, 2253-2254.
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J. AM. CHEM. SOC. 2004, 126, 9326-9329
10.1021/ja048554f CCC: $27.50 © 2004 American Chemical Society

Dialkyl Nitroxides as Probes for the Exchange of Solutes
A R T I C L E S
Table 1. EPR Spectral Parameters in Water and in MPC (Bold)
for Nitroxides 2-6; Equilibrium (Keq) and Rate (k+, k-) Constants
for the Complexation Process
probe
a(N)a
a(2H )a
g-factor
K
a (M-1
eq
)
k
+ b (M-1 s-1
)
k- b (s-1
)
â
2
3
16.17
16.17
15.71
16.25
15.81
16.14
15.82
16.32
15.70
10.26
10.18
9.21
10.38
9.23
10.16
9.19
10.35
9.00
2.0056
2.0056
2.0057
2.0056
2.0057
2.0056
2.0057
2.0056
2.0057
4178
7.8 × 109
8.0 × 109
7.7 × 109
8.2 × 109
2.1 × 106
7.4 × 105
1.9 × 106
4.9 × 105
4
5
6
11 250
5683
18 374
Figure 1. EPR spectra of 5 recorded in water (a) and in the presence of
MPC-C8-TEG 0.3 mM (b) and computer simulation of the latter one (c).
a T ) 298 K. b T ) 310 K.
Scheme 1
On the contrary, EPR spectra of nitroxides containing para-
alkyl substituents in the phenyl ring (3-6) recorded in the
presence of MPC-C8-TEG showed additional signals (see Figure
1b). When the concentration of MPC-C8-TEG nanoparticle was
increased up to 1.3 mM, the spectrum of the new species became
dominant, allowing the determination of its spectral parameters
(see Table 1).
This species was identified as the radical hosted in the MPC
monolayer, in equilibrium with the free nitroxide (Scheme 2),
on the basis of the following experimental evidence.
temperature, indicating that the lifetime of nitroxides in the
associated and free form is comparable to the EPR time scale;
this enabled us to measure the rate constants for the association
and dissociation processes. In this paper, we describe the use
of EPR to address the specific question of the interaction of an
hydrophobic probe with the monolayer of water-soluble Au-
MPC (MPC-C8-TEG) and how it compares with that of
micelles. The monolayer of these nanoparticles was made by
thiol 1 (see Scheme 1) comprising a short alkyl chain (C7) close
to the gold surface and a triethylene glycol monomethyl ether
unit (TEG).6c
(i) The EPR nitrogen hyperfine splitting a(N) was signifi-
cantly smaller than that of the free radical (see Table 1), this
indicating that the nitroxide group is located in a less polar
environment shielded from the aqueous solvent. The reduction
of a(N), remaining approximately constant by increasing the
length of the linear alkyl chain, is due to the larger weight of
the nitroxide mesomeric forms in which the unpaired electron
is localized on the oxygen rather than on the nitrogen atom.
This indicates that the polarity of the environment surrounding
the nitroxide function is similar for all radicals 3-6 and that
the N-O group is deeply inside the monolayer with the polar
O-H group close to the hydrophilic region.
(ii) The high-field EPR lines of the new species were
characterized by a lower height, due to the slower motion of
the radical probe when hosted in the monolayer, resulting in
incomplete averaging of the anisotropic components of the
hyperfine and g-tensors.
(iii) The molar ratio between the complexed and free radical,
obtained from the EPR spectra, increased linearly with increas-
ing concentration of the dissolved MPC-C8-TEG. Addition of
some water to the most concentrated solution in MPC-C8-TEG
(1.3 mM) resulted in an EPR spectrum consisting only of the
signals due to the unbound species, this providing experimental
evidence that the EPR signals observed in the presence of MPC-
C8-TEG are due to the radical hosted in the MPC monolayer
in equilibrium with the free nitroxide.
It should be remarked that under the above experimental
conditions the nitroxide is stable in the presence of Au-MPC
during EPR measurements. In the literature, it has been
reported7,11 that adsorption of nitroxide radicals onto a Au
particle surface lead to the loss of its EPR signal. This behavior
has been attributed to exchange interactions between the
unpaired electron and conduction-band electrons of the metal.
Our results clearly indicate that protection of a Au nanoparticle
with organic monolayer avoids a direct contact between the
Results and Discussion
The radical probes we have used in this study belong to a
family of para-substituted benzyl hydroxyalkyl nitroxides
(2-6, Scheme 1). They were generated directly inside an EPR
tube by oxidizing the parent amine (1 mM) with Oxone (1 mM)
in the presence of variable amounts of MPC-C8-TEG. The
amine precursors were prepared by reacting 2-methylalanine
with the appropriately substituted benzoyl chlorides followed
by reduction with 2 equiv of LiAlH4 (see Supporting Informa-
tion). The stability, under oxidative conditions, of MPC-C8-
TEG nanoparticles was checked spectrophotometrically after the
addition to a water solution of nanoparticles of a larger excess
of Oxone. After 5 min, no significative shifting of the surface-
plasmon band of the MPC-C8-TEG in the UV spectrum was
observed, indicating that MPC are stable for the time required
for recording an EPR spectrum.
Good EPR spectra of nitroxides 2-6 were obtained by
oxidation of the corresponding amines (1 mM) with Oxone
(1 mM) in water at 298 K. As an example, in Figure 1a is
reported the spectrum obtained with nitroxide 5. All the spectra
were straightforwardly interpreted on the basis of the coupling
of the unpaired electron with nitrogen and with the two equiv-
alent benzylic protons, the spectroscopic parameters being
reported in Table 1. When the EPR spectrum of nitroxide 2 is
recorded in the presence of MPC-C8-TEG, no additional lines
beside those due to the nitroxide radical in water solution were
observed, this suggesting negligible affinity of 2 for the
monolayer of MPC-C8-TEG.
(11) Barkley, P. G.; Hornak, J. P.; Freed, J. H. J. Phys. Chem. 1986, 84, 1886-
1900.
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A R T I C L E S
Lucarini et al.
Scheme 2
nitroxide function and the metal surface due to the location of
the spin probe inside the monolayer.
procedures based on the density matrix theory13 and assuming
a two-jump model as illustrated in Scheme 2, led to the
determination of the rate constants k+ and k- for the association
and dissociation processes, respectively (see Table 1).
Since the MPC-C8-TEG nanoparticles were present in large
excess with respect to the radical species,12 whose concentration
was in the range (1-2) × 10-5 M in all experiments, the
association constant, Keq, was obtained from the following
equation: Keq ) xMPC/(xwater × [MPC]). Here [MPC] denotes
the initial concentration of MPC-C8-TEG nanoparticles, and
xMPC and xwater are the mole fractions of the complexed and
free radical species, respectively, determined by simulation of
the EPR spectra (see Figure 1c). The values of Keq (see Table
1) indicate that the solubilization of the probes by MPC-C8-
TEG is clearly dependent on the hydrocarbon chain length: Keq
increases significantly by increasing the probe lipophilicity, with
the exception of the bulky tert-butyl group.
The dependence of these two rate constants on the nature of
the p-alkyl group of the substrate was quite different, k+ being
almost independent of it, while k- decreased significantly by
increasing the lipophilicity of the probe, with the exception of
the tert-butyl group. For the association process, both the
independence on the R group and the high value of k+ seem to
indicate that rate of solubilization is controlled by diffusion. In
the present case, where diffusion of a small molecule to a
relatively larger sphere occurs, the process can be considered
as a sequence of jumps over free-energy barriers between
adjacent equilibrium positions in the liquid, so that the following
equation can be derived: k+ ) 4πNRMPCDprobe, where D is the
diffusion coefficient of the probe in water, and R is the
hydrodynamic radius of MPC-C8-TEG.14 Using Dprobe ≈ 7 ×
10-6 cm2/s15 and RMPC ) 41 Å (see Experimental Section), one
obtains k+ ≈ 2.2 × 1010 M-1 s-1, a value significantly higher
than those experimentally measured for nitroxides 3-6
(≈ 8.0 × 109 M-1 s-1). Actually, the energy barrier to the
transfer of the probe from the aqueous phase to the interior of
the monolayer can be imagined to reflect the search for a suitable
spot on the surface among hydrophilic TEG chains and hydration
water, a difficult process for nitroxides 3-6 containing a polar
OH group.
The selective line-broadening, observed when recording the
EPR spectra of 3-6 in the presence of protected gold nano-
particles at 310 K (see Figure 2), indicates rapid exchange of
the nitroxide between the aqueous phase and the monolayer,
which modulates the nitrogen and proton hyperfine splittings
and, to a smaller extent, the Zeeman interaction. At 333 K, the
observed splittings are completely averaged over the residence
times in the two environments, in accordance with a fast
exchange process in the EPR time scale. Simulation of the
exchange-broadened EPR spectra, by using well-established
The values of the dissociation rate constant, k-, indicate that
the escape parameters of a given probe are determined by the
hydrophobicity of its alkyl chain, similarly to what was found
in micelles.10e Comparison of the values found when R ) n- or
tert-butyl indicates that for similar hydrophobicities an increased
bulk reduces the residence time in the monolayer.
Conclusions
In conclusion, we have determined for the first time, using
EPR spectroscopy, the partition isotherms and exchange rates
of a radical probe between an aqueous solution and the
monolayer of water-soluble Au-MPC. In comparison to micelles
where it is impossible to know precisely how the monomers
(12) Under these conditions, the nitroxides/MPC ratio was low enough to avoid
possible anomalous effects due to multiple occupancy of MPC.
(13) (a) Freed, J. H.; Fraenkel, G. K. J. Chem. Phys. 1963, 39, 326-348. (b)
Hudson, A.; Luckhurst, G. R. Chem. ReV. 1969, 69, 191-225.
(14) Rharbi, Y.; Li, M.; Winnik, M. A.; Hahn, K. G. J. Am. Chem. Soc. 2000,
122, 6242-6251.
(15) Substituted aromatic compounds have D values in the range 5-9 × 10-6
cm2/s. Burkey, T. J.; Griller, D.; Lindsay, D. A.; Scaiano, J. C. J. Am.
Chem. Soc. 1984, 106, 1983-1985.
Figure 2. EPR spectra of nitroxide 6 recorded in the presence of MPC-
C8-TEG 0.3 mM at different temperatures.
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Dialkyl Nitroxides as Probes for the Exchange of Solutes
A R T I C L E S
amorphous carbon film. The grid was then dried in air for 24 h. Images
were obtained with a JEOL 3010 high-resolution electron microscope
(1.7 nm point-to-point) operating at 300 keV using a Gatan slow-scan
CCD camera (mod.794). Diameters were measured manually using a
Gatan software Digital Micrograph (ver. 3.4.1) on at least 100 particles.
The MPC used have an average core diameter of 3.4 ( 0.7 nm and
a calculated MW of 324 700 Da. When a completely extended ligand
is considered, the calculated MPC radius is about 4.1 nm.
aggregate due to the disorder of the system, the monolayer of
MPC-C8-TEG shows well-defined regions due to the specific
orientation of the constituent thiols. This has allowed us to
identify with confidence the locations of the probes in the
monolayer at the boundary between the hydrocarbon and
polyether regions, this being due to their amphiphilic character.
Partition isotherms and rate of the exchange are nevertheless
governed by the hydrophobic portions of the probes. Finally,
EPR turned out to be a suitable technique to investigate the
kinetic behavior of guest radicals in MPC, since the character-
istic lifetime of the probes in the monolayer is comparable to
the EPR time scale.
EPR Measurements. Radicals 2-6 were generated by mixing 1
µL of a methanol solution containing the corresponding amine
(0.1 M) and 1 µL of a water solution containing Oxone (0.1 M) with
100 µL of a water solution containing variable amounts of MPC-C8-
TEG. Samples were transferred into capillary tubes (1 mm i.d.) and
then placed inside the thermostated cavity of an EPR spectrometer.
EPR spectra were obtained using a Bruker ESP300 spectrometer
equipped with an NMR gaussmeter for field calibration and a Hewlett-
Packard 5350B microwave frequency counter for the determination of
the g-factors, which were referenced to that of the perylene radical
cation in concentrated H2SO4 (g ) 2.002 58). The sample temperature
was controlled with a standard variable temperature accessory and was
monitored before and after each run using a copper-constantan
thermocouple. The instrument settings were as follows: microwave
power 5.0 mW, modulation amplitude 0.05 mT, modulation frequency
100 kHz, scan time 180 s. Digitized EPR spectra were transferred to a
personal computer for analysis using digital simulations carried out
with a program developed in our laboratory and based on a Monte
Carlo procedure.10a The input data for the program are the number of
nonequivalent nuclei, the hyperfine splitting constants of the free and
included radical, the intrinsic line width in the absence of exchange,
and the rate constants for the exchange process.
Experimental Section
General. NMR spectra involving nanoparticles were taken in CDCl3
that had been stirred over K2CO3 for at least 24 h prior to use.
ω-Bromooctanoic acid and thiolacetic acid were purchased from
Aldrich, thiolacetic acid was distilled prior to use, tri(ethylene glycol)
monomethyl ether was purchased from Fluka and used without further
purification. Dry solvents were obtained from Fluka. All other solvents
were reagent grade and used as received.
Synthesis of MPC-C8-TEG. HAuCl4‚3H2O (59.2 mg, 0.15 mmol)
and 30 mL of milliQ water were introduced in a 250 mL round-bottom
flask, and to the pale yellow solution thiol 16c (32 mg, 0.1 mmol)
dissolved in 30 mL of deoxygenated methanol was added. Upon
addition of the thiol, the solution turned reddish-brown, and the mixture
was allowed to stir 30 min at room temperature and for a further 30
min at 0 °C. To the solution NaBH4 (66 mg, 1.7 mmol) in 15 mL of
water was added in 3 min and 30 s. The reaction mixture turned
immediately wine-red; it was stirred at 0 °C for 30 min and at room
temperature for 2 h. The solvent was removed under reduced pressure
without heating above 35 °C. The residue was dried in vacuo, then
dissolved in dichloromethane, and centrifuged. The solution was
concentrated to a small volume and treated with ether in order to
precipitate the nanoparticles. The black solid was washed copiously
with ether and purified by gel permeation chromatography (Sephadex
LH-60, methanol). After this treatment, 35 mg of nanoparticles were
obtained. UV-vis (water) λmax (nm); a (l g-1 cm-1) 510.4; 8.2. IR (KBr)
ν (cm-1) 3296, 2915, 2848, 1736, 1642, 1548, 1465, 1452. 1351, 1258,
1197, 1107, 1019, 1003, 798. 1H NMR (250 MHz, CDCl3) δ 3.63 (br),
3.54 (br), 3.38 (br), 2.19 (br), 1.61 (br), 1.33 (br). 1H NMR (250 MHz,
D2O) δ 3.50 (br), 3.22 (br), 2.10 (br), 1.44 (br), 1.07.
Acknowledgment. Financial support from MIUR, Contract
2002038342 (P.F., M.L., and G.F.P.) and from the University
of Padova (progetti di Ricerca di Ateneo 2000 and Progetti
Giovani Ricercatori 2001) and MIUR, Contract 2003054199 (to
L.P.) are gratefully acknowledged.
Supporting Information Available: General procedure for
the preparation of probes 2-6, some representative EPR spectra
of 3-6, detailed radical ratios determined from the EPR spectra,
and stability experiments of MPC-C8-TEG in the presence of
Oxone. This material is available free of charge via the Internet
at http://pubs.acs.org.
TEM samples were prepared by placing a single drop of <1 mg/
mL dichloromethane solution onto a 200-mesh copper grid coated with
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