An EPR method for measuring the rate of distribution of organic substrates between cyclodextrin, micelles and water

Article abstract of DOI:10.1039/b718049g

The combined use of selected nitroxides and EPR spectroscopy has been proved to be suitable for studying the partitioning rate of a given substrate in cyclodextrin-micelle systems. The Royal Society of Chemistry.

Full text of DOI:10.1039/b718049g

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An EPR method for measuring the rate of distribution of organic
substrates between cyclodextrin, micelles and waterw
a
a
b
b
Elisabetta Mileo, Paola Franchi, Roberto Gotti, Claudia Bendazzoli,
a
a
Elisabetta Mezzina and Marco Lucarini*
Received (in Cambridge, UK) 22nd November 2007, Accepted 20th December 2007
First published as an Advance Article on the web 23rd January 2008
DOI: 10.1039/b718049g
The combined use of selected nitroxides and EPR spectroscopy
has been proved to be suitable for studying the partitioning rate
of a given substrate in cyclodextrin–micelle systems.
significantly augmenting the arsenal of methods available for
studying the partitioning of a given substrate in different
pseudo-phases.
The method is based on the significant differences in the
EPR parameters shown by tert-butyl benzyl nitroxide (TBBN)
Mixed organized media represent an intriguing system because
of the possibility for a given guest to interact selectively with a
1
single binding site. When a substrate is added to such system
8
9
when it experiences water, cyclodextrin cavity or micellar
10
environments (see Table 1). The partitioning of nitroxide
the noncovalent binding of the guest (G) with the different
hosts (H) to form a guest–host complex (C) is observed:
probes in the hydrophobic environment of SDS micelle gives
rise to a reduction of the value of both nitrogen and b-protons
splittings, with the result that the resonance fields for the
kON
M (2H ) = ꢁ1 lines of the free and included species are
ꢀ
!
I
b
G þH ꢀC
ð1Þ
kOFF
significantly different from those of the nitroxide dissolved in
water. These differences are even more pronounced when
TBBN is included in the cavity of CD’s due to both polar
and conformational changes occurring upon complexation.
The EPR spectra also show a strong linewidth dependence
on temperature both in the presence of SDS micelle and CD,
indicating that the lifetime of the radical in the associated and
free form is comparable to the EPR timescale. Because of this
favorable feature, the analysis of the line shape makes it
possible to measure the rate constants for the partition of
the probe in the pseudo-phases.
where kON and kOFF are the rate constants of complex
formation and dissociation, respectively. The stability of the
complex is generally described in terms of the equilibrium
dissociation constant, K = kOFF/kON. In order to understand
d
the chemistry of this system, it is vital to know the rate at
which an organic molecule is partitioned between the different
2
phases.
One of the most studied system is the cyclodextrin–micelle
mixed system, because of its application in separation-based
3
affinity methods (i.e. capillary electrophoresis), as a reaction
The EPR spectra at 298 K of TTBN, produced by reaction
of the magnesium salt of monoperoxyphthalic acid with tert-
butyl benzyl amine in the presence of heptakis-(2,6-O-di-
methyl)-CD (DM-b-CD) 5.3 mM or SDS 33 mM are shown
in Fig. 1a and 1b, respectively. The spectra show in both cases
two sets of signals due to the radical in the aqueous phase and
to that included in the CD cavity (Fig. 1a) or solubilised in the
micellar pseudo-phase (Fig. 1b). In the presence of a mixture
of CD and SDS at the same concentration as above, the EPR
spectrum differs significantly from the previous ones (see
ESIw). All the spectra can be correctly reproduced only by
assuming the kinetic scheme reported in Scheme 1 in which the
radical probe is exchanging, with a rate comparable to the
EPR time scale, between the water phase and both the CD
cavity and the micellar pseudo-phase.
4
medium and in supramolecular devices. While rates of
5
6
solubilization into surfactant solutions and of the inclusion
7
complexation by cyclodextrins (CDs) of specific probes have
been extensively investigated using a variety of methods, the
direct measure of individual binding events between analyte
molecules and a mixture of both CD and micelle is still
challenging.
EPR spectroscopy is characterised by a peculiar timescale
which is comparable to that of many complexation processes.
Here we introduce and describe an EPR method that allows,
for the first time, direct and simultaneous measurement of the
concentration of an organic spin probe in three different
‘
‘pseudo-phases’’ (namely SDS micelles, CDs and water), if
it exceeds the limit of detection. This new method allows the
determination of all thermodynamic and kinetic parameters,
K , kON, and kOFF, in a single experiment that requires only a
d
minute amount of substrates. This has the potential for
Table 1 EPR parameters of tert-butyl benzyl nitroxide (1 G =
0.1 mT)
a
Department of Organic Chemistry ‘‘A. Mangini’’, Via S. Giacomo
11-40126 Bologna. E-mail: marco.lucarini@unibo.it
Department of Pharmaceutical Sciences, Via Belmeloro 6-40126
Bologna
b
a(N)/G
a(2H
b
)/G
g-factor
Water
SDS
DM-b-CD
16.69
16.04
15.60
10.57
8.84
7.80
2.0056
2.0057
2.0058
w Electronic supplementary information (ESI) available: EPR spectra
of TBBN recorded in the presence of different amounts of CD and
SDS. See DOI: 10.1039/b718049g
This journal is ꢂc The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1311–1313 | 1311

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Scheme 1
SDS] r [CD]: Under this condition the amount of radical
[
included in the CD cavity decreases proportionally to the
amount of surfactant present in the solution. This effect is
attributed to complexation of the surfactant monomer by CD
and release of the probe into the bulk aqueous medium giving
rise to an increase in the residence time of the probe in the
water phase (entries 3–4). According to the fact that in this
condition the free dissolved SDS monomer concentration is
well below the critical micelle concentration (cmc) no EPR
signals due to the radical partitioned in the SDS phase are
observed.
[SDS] 4 [CD]: The formation of SDS–CD complex in-
Fig. 1 EPR spectra of TBBN recorded in water at 298 K. (a) DM-b-
CD 5.3 mM; (b) SDS 33 mM; (c) b-CD 16 mM and SDS 49 mM; (d)
DM-b-CD 16 mM and SDS 49 mM. (e) Theoretical simulation of
spectrum (d) obtained with the rate constant reported in Table 1, entry 11.
creases the concentration of surfactant required for micelliza-
12
tion, and the critical micelle concentration of a micellar
system in the presence of a cyclodextrin (cmcapp) is equivalent
to the combined concentrations of surfactant monomers com-
plexed to the CD, and of free dissolved monomer in equili-
Simulation of the exchange-broadened EPR spectra, by
using well established procedures based on the density matrix
brium with the micellized surfactant, cmcreal
.
Once
1
theory and assuming a three-jump model as illustrated in
1
micellization starts, the system behaves like a typical micellar
system. Analysis of EPR data shows that this is actually the
case when b-CD (see Entries 5–8) is employed as the macro-
cyclic host. As an example Fig. 1c shows the EPR spectrum
recorded in the presence of SDS 49 mM and b-CD 16 mM
which perfectly matches that one recorded in the presence of
SDS 33 mM (Fig. 1b).
Scheme 1, led to the determination of the residence time (t ) of
X
the paramagnetic species in the three different pseudo-phases
which are related to rate constants (see Table 2) of the
exchange processes by the following expressions:
CD
MIC
CD
MIC
t
CD = 1/kOFF; tMIC = 1/kOFF; tH O = 1/kON + 1/kON (2)
2
A change in the nature of the macrocyclic host has, how-
ever, a dramatic effect on the partitioning behaviour of the
radical probe. In the presence of a small amount of methylated
cyclodextrins (DM-b-CD or TM-b-CD) the EPR spectra are
characterised by an increased broadening of the external lines,
indicating that the exchange rate of the probe between water
and the micellar pseudo-phase is becoming faster (see entries
9–10). Further addition of methylated cyclodextrins in the
solution results in a dramatic decrease of the life time of the
probe in the SDS micelles. Nevertheless, when the
By recording the corresponding EPR spectra it is possible,
therefore, to obtain the distribution of the radical probe in the
different environments at different concentrations of surfac-
tant and cyclodextrin. As an example, in Fig. 2 is reported the
variation of the molar fraction of TBBN partitioned in the
micellar phase in the presence of different DM-b-CD/SDS
concentrations.
Depending on the relative amount of CD and SDS we can
distinguish different regimes:
Table 2 Selected EPR rate constants at 298 K for the partition of TBBN in the micellar and CD locations
CD ꢀ1
ON/s
CD
ꢀ1
MIC ꢀ1
ON /s
MIC ꢀ1
kOFF/s
Entry
[SDS]/mM
Cyclodextrin (mM)
k
k
OFF/s
k
6
6
6
5
5
5
5
5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
a
—
—
3.4
5.0
33
26
33
49
33
33
49
105
73
53
b-CD (5.3)
3.6 ꢃ10
3.4 ꢃ10
1.7 ꢃ10
3.4 ꢃ10
—
5.3 ꢃ10
6.1 ꢃ10
6.5 ꢃ10
7.0 ꢃ10
—
—
—
—
—
—
—
—
—
DM-b-CD (5.3)
DM-b-CD (5.3)
DM-b-CD (5.3)
—
b-CD (16)
b-CD (16)
6
5
6
6
6
7
7
7
7
7
6
6
6
6
6
7
7
7
7
7
6.2 ꢃ10
8.0 ꢃ10
3.0 ꢃ10
6.2 ꢃ10
5.5 ꢃ10
1.6 ꢃ10
1.5 ꢃ10
1.5 ꢃ10
2.2 ꢃ10
1.5 ꢃ10
3.9 ꢃ10
3.9 ꢃ10
3.9 ꢃ10
3.9 ꢃ10
7.8 ꢃ10
4.7 ꢃ10
2.8 ꢃ10
1.0 ꢃ10
2.8 ꢃ10
2.4 ꢃ10
4
5 a
5 a
5 a
5 b
5 b
5 b
5 b
5 b
6 ꢃ10
5.3 ꢃ10
5.3 ꢃ10
5.3 ꢃ10
6.1 ꢃ10
6.1 ꢃ10
6.1 ꢃ10
6.1 ꢃ10
6.1 ꢃ10
—
4
4
4
o3 ꢃ10
o3 ꢃ10
o4 ꢃ10
b-CD (16)
DM-b-CD (5.3)
DM-b-CD (20)
DM-b-CD (16)
DM-b-CD (20)
DM-b-CD (40)
TM-b-CD (20)
b
4
0
1
2
3
4
8.0 ꢃ10
4.0 ꢃ10
{4 ꢃ10
4
4
4
8.0 ꢃ10
—
Assumed equal to that of entry 1. Assumed equal to that of entry 2.
1
312 | Chem. Commun., 2008, 1311–1313
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Fig. 2 Molar fraction of TBBN partitioned in SDS micelles in the
presence of different DM-b-CD/SDS concentrations.
Fig. 3 Mobility data of tert-butyl benzyl ketone as a function of the
SDS concentration and the nature of cyclodextrin employed.
concentration of DM-b-CD is so much so that ([CD] +
cmcreal) 4 [SDS], the EPR spectrum can be correctly simu-
lated by admitting that a sizeable fraction of the probe is still
experiencing a hydrophobic environment. While the dramatic
reduction of the residence time of the radical guest in the micelle
suggests that the micellar structure is altered significantly in the
presence of methylated cyclodextrins, the existence of radicals
dissolved in an hydrophobic aggregate for concentration of SDS
below cmcapp (cmcapp = cmcreal + CD) indicate that this altered
micelle is still able to solubilise the probe. According to recent
found with b-CD, in the presence of SDS between 20 and 28
mM, that is below the hypothetical cmcapp ([DM-b-CD] +
cmcreal = 28 mM) the transport ability of the micellar carrier
is still maintained with DM-b-CD, this being an indication
that a modified micellar carrier is present in the solution as
predicted by EPR measurements. Work is in progress to
investigate the effect of methylated CDs on the CE-based
separation of enantiomers when the SDS concentration is in
the premicellar range.
In conclusion, the combined use of selected nitroxide and
EPR spectroscopy has been proved to be suitable for studying
the partitioning rate of a given substrate in CD–micelle
systems. On the condition that the spectroscopic parameters
of the probe are sufficiently different to distinguish the differ-
ent environment experienced by the radical, EPR data can be
employed to predict the partitioning behaviour of non radical
analytes in mixed organised systems. We foresee the potential
role of EPR in extending the utility of this technique by using
13
findings by Dreiss and coworkers we can suppose that the
presence of methylated cyclodextrins results in a lowering of the
aggregation number, and in an increase of solvent penetration
and polydispersivity of the micelle.
From an applicative point of view we checked if the
residence time of the probe in each environment determined
by EPR could be employed to predict the electrophoretic
behaviour of a given analyte. Capillary electrophoretic (CE)
analysis of neutral solutes in CD-micellar systems is based on
the differences in the mobility of the analytes in the homo-
geneous phase. The anionic SDS micelle acts as a carrier and
the inclusion of the solutes into the neutral CD cavity is a
process in competition with the partitioning into the micelle.
When the residence time of the probe in the cyclodextrin is
long compared with the residence time in the micelle, the
mobility of the neutral probe approaches to zero; this condi-
tion is also observed when surfactant concentration is below
the critical micelle concentration.
1
4
probes characterized by a different lipophilicity or contain-
5
ing a chiral centre.
1
Notes and references
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9
L. Garcıa-Rıo, J. R. Leis, J. C. Mejuto and J. Perez-Juste, J. Phys.
CE experiments were performed by analyzing the electro-
phoretic behaviour of tert-butyl benzyl ketone, the diamag-
netic analogue of TBBN, in the presence of either b-CD and
DM-b-CD at 16 and 20 mM, respectively. SDS was supplemen-
ted as a micelle separation carrier in a wide concentration range
´
Chem. B, 1998, 102, 4581.
´
´
Y. Suzaki, T. Taira, D. Takeuchi and K. Osakada, Org. Lett.,
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(
e
15–165 mM) and the effective electrophoretic mobility (m ) of
the probe was plotted against the SDS concentration (Fig. 3).
As expected, in the absence of cyclodextrin only small
mobility variations were observed in the investigated SDS
range, while, in the presence of b-CD, the mobility variation
profile shows a marked break point which corresponds to a
1
0 G. Brigati, P. Franchi, M. Lucarini, G. F. Pedulli and L. Valgi-
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1
1
1 A. Hudson and G. R. Luckhurst, Chem. Rev., 1969, 69, 191.
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´ ´ ´
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´
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=
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2
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1
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the carbonyl probe is significantly reduced. According to EPR
data this should be attributed to the reduced residence time of
the probe in the micellar pseudo-phase. Conversely to that
1
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This journal is ꢂc The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1311–1313 | 1313