Vesicular catalysis of the decarboxylation of 6-nitrobenzisoxazole-3- carboxylate. The effects of sugars, long-tailed sugars, cholesterol and alcohol additives

Article abstract of DOI:10.1002/poc.1028

The effects of the addition of sugars, long-tailed n-alkyl pyranosides, n-alkyl glycerol ethers and n-alcohols on the properties of di-n- hexadecyldimethylammonium bromide (DHAB) vesicles have been studied. Properties that were examined include the stability, morphology, phase of the tails, and catalytic rate acceleration of the unimolecular decarboxylation of the 6-nitrobenzisoxazole-3-carboxylate anion (6-NBIC). The kinetic data were analyzed on the basis of the pseudophase model and show a rate acceleration of a factor of about 1000 relative to the reaction in water. Upon addition of most additives an inhibiting effect on the decarboxylation reaction of 6-NBIC is observed relative to the reaction in vesicles without any additive. The largest inhibition was observed in the case of cholesterol. Contrastingly, n-dodecyl-β-maltoside (in the spherical vesicle region) and trehalose accelerate the reaction. The activation parameters show that the most significant contribution to the Gibbs energy of activation is the enthalpic factor, with a partly compensating entropic contribution. Copyright

Full text of DOI:10.1002/poc.1028

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2006; 19: 249–256
Published online 24 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.1028
Vesicular catalysis of the decarboxylation of
6-nitrobenzisoxazole-3-carboxylate. The effects of
sugars, long-tailed sugars, cholesterol and alcohol
additives
*
Mahthild G. M. Jongejan, Jaap E. Klijn and Jan. B. F. N. Engberts
Contribution from the Physical Organic Chemistry Unit, Stratingh Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands
Received 9 November 2005; revised 16 January 2006; accepted 17 January 2006
ABSTRACT: The effects of the addition of sugars, long-tailed n-alkyl pyranosides, n-alkyl glycerol ethers and n-
alcohols on the properties of di-n-hexadecyldimethylammonium bromide (DHAB) vesicles have been studied.
Properties that were examined include the stability, morphology, phase of the tails, and catalytic rate acceleration of
the unimolecular decarboxylation of the 6-nitrobenzisoxazole-3-carboxylate anion (6-NBIC). The kinetic data were
analyzed on the basis of the pseudophase model and show a rate acceleration of a factor of about 1000 relative to the
reaction in water. Upon addition of most additives an inhibiting effect on the decarboxylation reaction of 6-NBIC is
observed relative to the reaction in vesicles without any additive. The largest inhibition was observed in the case of
cholesterol. Contrastingly, n-dodecyl-b-maltoside (in the spherical vesicle region) and trehalose accelerate the
reaction. The activation parameters show that the most significant contribution to the Gibbs energy of activation
is the enthalpic factor, with a partly compensating entropic contribution. Copyright # 2006 John Wiley & Sons, Ltd.
KEYWORDS: 6-NBIC; DHAB; vesicles; vesicular catalysis
INTRODUCTION
strongly on the nature of the alkyl tail. In the case of the
addition of n-alkyl pyranosides it was concluded that
partial dehydration of small ions present at the interface
takes place. Concomitantly, the local water concentration
is reduced. However, for bimolecular reactions the
reactivity and distribution of both reactants have to be
considered, often leading to difficulties in interpretation
of the experimental data. Alternatively, when a unim-
olecular reaction is used these problems do not play a role,
therefore the unimolecular decarboxylation reaction of
the 6-nitrobenzisoxazole-3-carboxylate anion (6-NBIC,
Scheme 1) was selected as a suitable reaction, because
this reaction is also very sensitive to changes in the local
polarity at the binding site(s) of the reactant.3–6 Apolar
solvents result in the formation of ion pairs, thereby
reducing the rate constant for the decarboxylation
reaction. Increasing the polarity will therefore increase
the rate constant compared to apolar solvents. Hydrogen
bonding, however, leads to a decrease in the rate constant
because of dominant hydrogen bond stabilization of the
initial state. Cationic vesicles accelerate this decarbox-
ylation reaction relative to pure water by a factor of ca.
1000.6 It has been shown that such vesicles can accelerate
the 6-NBIC reaction by approximately 30% compared to
cationic micelles.7 Synthetic cationic vesicles are also
4–15times more efficient for the decarboxylation than
phospholipid vesicles.8 The interaction between the
The membrane is an essential part of the biological cell by
aiding replication, compartmentalizing the cell contents
and controlling the flux of small molecules into and out of
the cell.1 Vesicles formed from (mixtures of) phospho-
lipids and/or synthetic amphiphiles have been used as
mimics for such membranes. Due to the often complex
structure of biomolecules, little is known about interac-
tions on a molecular scale between the various
components of biological membranes.2 Previously,
reaction kinetic measurements of bimolecular reactions
have been employed in attempts to understand some of
these interactions in model membranes composed of
more than one amphiphilic component. In these systems,
besides one or more types of amphiphile (which form
vesicles on their own), other amphiphilic molecules such
as long-tailed alcohols and n-alkyl pyranosides were
added in various concentrations. It was found that the
direction and extent of the effect on the vesicular
rate constant and the binding constant of the organic
substrate upon the addition of long-tailed alcohols depend
*Correspondence to: J. B. F. N. Engberts, Contribution from the
Physical Organic Chemistry Unit, Stratingh Institute, University of
Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
E-mail: J.B.F.N.Engberts@rug.nl; or engberts@chem.rug.nl
Contract/grant sponsor: NRSC-C.
Copyright # 2006 John Wiley & Sons, Ltd.
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250
M. G. M. JONGEJAN, J. E. KLIJN AND J. B. F. N. ENGBERTS
-
-
hexadecanol (HD). Class B contains two glycerol ethers
O
O
O
O
(with one or two alkyl tails) and cholesterol. Class C
includes C12-tailed surfactants with sugar headgroups.
Class D contains sugars without a hydrophobic anchor:
glucose (Glu), maltose (Mal) and trehalose (Treh). In
addition to kinetic measurements, differential scanning
calorimetry (DSC) and cryo-transmission electron micro-
scopy (cryo-TEM) measurements have been carried out.
CN
O-
N
N
O
O
O2N
O2N
O2N
+ CO2
Scheme 1. Decarboxylation reaction of 6-nitrobenzisoxa-
zole-3-carboxylate (6-NBIC)
cationic head group and the 6-NBIC anion promotes
binding at the vesicular interface, thereby decreasing the
number of hydrogen bonding opportunities between
water and the substrate, with a concomitant increase of
the rate constant. It is thus expected that the additives will
affect the local environment of the substrate binding sites,
resulting in changes in the vesicular rate constants.
Di-n-hexadecyldimethylammonium bromide (DHAB)
was selected as the vesicle-forming amphiphile. Four
classes of additives (Scheme 2) were chosen, with
systematic changes in their structure in order to gain an
understanding of the effect of each component on the
unimolecular decarboxylation reaction. Class A includes
the monohydric alcohols n-dodecanol (DD) and n-
EXPERIMENTAL
Di-n-hexadecyldimethylammonium bromide (DHAB ꢀ
97%), n-hexadecanol (ꢀ99%) and n-dodecyl-b-D-gluco-
pyranoside (ꢀ99%) (CMC ¼ 1.9 ꢁ 10ꢂ4 mol lꢂ1)9 were
obtained from Fluka. Cholesterol (ꢀ99%) was purchased
from Aldrich, acetonitrile (99%) was from Acros, D(þ)-
glucose (anhydrous) was bought from Merck and maltose
from Sigma. 1-O-n-Hexadecyl-rac-glycerol and 1,2-O-
di-n-hexadecyl-rac-glycerol were purchased from Brun-
schwig Chemie and stored at ꢂ208C. n-Dodecyl-b-
maltoside (>99.5%) (CMC ¼ 1.5 ꢁ 10ꢂ4 mol lꢂ1)9 was
purchased from Glycon and was also stored at ꢂ208C. All
chemicals were used without further purification.
+
Br-
N
DHAB
OH
DD
HD
class A
class B
OH
HO
HO
O
1 OHD
HO
O
1,2 OHD
O
OH
Chol
O H
O H
O H
O H
O
O
O
C12Glu
C12Mal
O H
O H
O H
O
H O
O
O H
H O
O
class C
H O
OH
OH
OH
OH
OH
class D
O
OH
OH
O
HO
HO
O
HO
HO
OH
OH
OH
O
O
OH
OH
O
HO
HO
HO
O
Glu
Mal
Treh
HO
OH
Scheme 2. Structure of DHAB (top) and the four classes of additives A–D: (A) DD is n-dodecanol and HD is n-hexadecanol; (B)
1-OHD is 1-O-n-hexadecyl-rac-glycerol, 1,2-OHD is 1,2-O-di-n-hexadecyl-rac-glycerol and Chol is cholesterol; (C) C12Glu is n-
dodecyl-b-D-glucopyranoside, and C12Mal is n-dodecyl-b-maltoside; (D) Glu is glucose, Mal is maltose and Treh is trehalose
Copyright # 2006 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2006; 19: 249–256

DECARBOXYLATION OF 6-NITROBENZISOXAZOLE-3-CARBOXYLATE
251
6-Nitrobenzisoxazole-3-carboxylate (6-NBIC) was
synthesized according to a literature procedure10,11 and
was stored at ꢂ208C.
in which kobs, kw and kves are the observed, aqueous and
vesicular rate constants, respectively; and K6-NBIC is the
binding constant of the organic substrate to the vesicular
surface.
In all experiments the minimum concentration of
DHAB was 9 mM, which is above the CVC (critical
vesicle concentration) of DHAB.13 At these concentra-
tions the 6-NBIC substrate is completely bound to the
vesicular interface owing to the relative excess of DHAB
and the high binding constant of 6-NBIC (>103 Mꢂ1).6
The strong binding originates mainly from electrostatic
interactions between the substrate and the oppositely
charged vesicles, but also from hydrophobic interactions.
Therefore, under these conditions it can be assumed that
Kinetic measurements
The vesicular solutions were prepared by weighing the
appropriate amounts of DHAB and additive and then
dissolving them in doubly distilled water. The samples were
left at 508C for 1 h and then sonicated at 508C for 15 min
(until a homogeneous, bluish solution was obtained).
The decarboxylation of 6-NBIC was monitored at
35.0 ꢃ 0.18C (which is above the main phase transition
temperature for DHAB vesicles of 288C12) by measuring
the increase in the absorption at 410 nm for at least five
half-lives of each of the samples. The samples contained
0.9 ml of DHAB þ additive, NaOH (100-fold excess of
6-NBIC) and a stock solution of 6-NBIC (4 ml of 6 mM
stock). Each of the kinetic measurements in the vesicular
solutions was repeated five times.
K6-NBIC[Amph] ꢅ 1 and kvesK6-NBIC[Amph] ꢅ kw. Con-
sequently, Eqn (1) can be simplified to:
kobs ¼ kves
(2)
In Fig. 1 the relative rate constants (kves/k0) as a
function of the mole fraction of additive are shown for the
different classes of additives. Rate constants for several
different mole fractions are reported in Table 1. As a
reference, the vesicular rate constant in the absence of
additive was taken to be k0. It is apparent that due to the
addition of most of the additives (class A and B) the rate
constant of the decarboxylation reaction decreases.
Figure 1(A) shows that the addition of n-dodecanol
(DD) has a larger inhibiting effect than the addition of
n-hexadecanol (HD). It has been shown previously that
n-decanol and n-hexadecanol in DHAB at 358C lead to a
similar decrease in the rate constant for the 6-NBIC
decarboxylation.12 However, at higher concentration the
reaction in the presence of n-decanol was slower than that
with hexadecanol. Under similar conditions the magni-
tudes of the vesicular rate constants for vesicles
containing HD were the same (within error), however
the effect of DD is more pronounced than that of
n-decanol. This would suggest that DD has a somewhat
larger perturbing effect on the vesicular interface.
Figure 1(B) compares the class B additives. Vesicles
containing the double-tailed glycerol ether 1,2-O-di-n-
hexadecyl-rac-glycerol (1,2-OHD) were less stable
compared to vesicles with other additives, therefore the
errors are larger in this case. However, 1,2-OHD seems to
decrease the rate constant more than the single-tailed
glycerol ether 1-O-n-hexadecyl-rac-glycerol (1-OHD).
Cholesterol (Chol) induces the largest inhibiting effect,
decreasing the vesicular rate constant by a factor of 3 at
50 mol.%. Figure 1(C) compares the kinetic effects of the
class C additives. It can be seen that n-dodecyl-b-
maltoside (C12Mal) does not follow the approximately
linear relationship between the natural logarithm of the
rate constant and mole fraction, as found for the other
additives. All points for C12Mal show a smaller decrease
Differential scanning microcalorimetry
All DSC measurements were carried out using a CSC
NANO II differential scanning microcalorimeter as
described previously.13 A total amphiphile concentration
of 2 mM and 2.25 mM sodium hydroxide were used for
the experiments. Five scans were taken between 5 and
1008C at a scan rate of 18C minꢂ1. The reference cell
contained doubly distilled water. The first scan was
neglected due to the thermal history of the machine, but
the other scans were all identical.
Cryo-electron microscopy measurements
Aliquots of a 5 mM amphiphile solution were deposited on
holey carbon grids in a controlled environmental chamber
at 358C and 100% humidity. The excess solution was
blotted off using filter paper and the samples were vitrified
by rapid plunging into liquid ethane. The micrographs were
recorded on a Philips CM 120 cryo-electron microscope
operating at 120 kV and using low-dose conditions.
RESULTS AND DISCUSSION
Decarboxylation of 6-NBIC
The rate constants for the unimolecular decarboxylation
of 6-NBIC were measured in the presence of DHAB
vesicles and analyzed using the pseudophase model
developed by Menger et al.14 According to the model the
observed rate constant of a unimolecular reaction can be
described by:
kw þ kvesK6ꢂNBIC½Amphꢄ
kobs
¼
(1)
1 þ K6ꢂNBIC½Amphꢄ
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252
M. G. M. JONGEJAN, J. E. KLIJN AND J. B. F. N. ENGBERTS
0.4
0.2
0.4
B
A
0.2
0.0
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
mole fraction additive
mole fraction additive
0.4
0.2
0.4
0.2
0.0
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
C
D
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
mole fraction additive
mole fraction additive
Figure 1. Plots of ln(kves/k0) versus the mole fraction of the additives in the DHAB vesicles: (A) the additives are HD (&) and DD
(&); (B) the additives are 1-OHD (~), 1,2-OHD (~) and Chol (x); (C) the additives are C12Glu (*) and C12Mal (*); (D) the
additives are Treh (ꢆ), Glu (^), and Mal (^). The lines are drawn to guide the eye. We note that HD, DD and Chol are above the
Tm at 30 mol.%
in the rate constant in comparison with n-dodecyl-b-
D-glucoside (C12Glu). Figure 1(D) compares the class D
additives and shows that the only unfunctionalized sugar
that affects the rate constant positively is trehalose (Treh),
but the effects are so small that no definite conclusions
can be drawn.
When the vesicular rate constants in the presence of
DD, C12Glu and C12Mal are compared, it can be
concluded that kves is highest for the sugar-based
additives. This slight increase in kves relative to vesicles
containing DD is probably a result of the (partial)
displacement of water from the Stern region by the sugar
moieties.15 Displacement of water molecules from the
Stern region has been shown for mixed micelles of
sodium n-dodecylsulfate and a sugar-based surfactant.16
Recently it was found, using reaction kinetic experiments,
that this is also the case for vesicles composed of C12Glu,
DHAB and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine).17 The 6-NBIC decarboxylation reac-
tion is sensitive towards hydrogen bonding. Hence,
if there are fewer possibilities for hydrogen bonding to
the organic substrate, the rate constant should increase.
However, considering that the vesicular rate constants
are lower compared to that for DHAB vesicles in the
absence of the additives, the beneficial effect on the
vesicular rate constants by the sugar groups is largely
counteracted by the effect of the tails of the sugar-based
additives. This is exemplified by the fact that DD has a
Table 1. Vesicular rate constants for all additives at
different mole percentages (all in liquid-crystalline state);
the value for pure DHAB vesicles is 8.6ꢁ10ꢂ3 sꢂ1
kves for different mol.% (ꢁ10ꢂ3 sꢂ1
)
Additive
10 mol.%
20 mol.%
30 mol.%
HD
DD
1-OHD
1,2-OHD
Chol
C12Glu
C12Mal
Treh
Glu
Mal
8.0
7.5
7.9
6.8
7.0
8.0
9.1
9.7
7.2
9.1
7.9
6.6
7.4
6.8
7.0
7.6
8.9
9.7
7.8
8.2
6.5
5.0
6.3
5.5a
4.2b
7.5
8.1
9.6
7.4
9.0
a Measured at 25 mol.%.
b Measured at 35 mol.%.
Copyright # 2006 John Wiley & Sons, Ltd.
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DECARBOXYLATION OF 6-NITROBENZISOXAZOLE-3-CARBOXYLATE
253
60
60
55
50
45
40
35
30
25
20
A
55
50
45
40
35
30
25
20
B
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.0
0.1
0.2
0.3
0.4
0.5
0.6
mole fraction additive
mole fraction additive
Figure 2. Main phase transition temperatures of DHAB vesicles in the presence of: (A) DD (&), HD (&), Chol (x), 1-OHD (~),
1,2-OHD (~); (B) C12Glu (*), C12Mal (*) and Treh (ꢆ). The horizontal lines represent the temperature at which the kinetic
experiments were carried out
larger effect on the vesicular rate than C12Glu and
C12Mal. Similarly, when vesicular solutions containing
HD and 1-OHD (class B) are compared, the vesicular rate
constants are slightly higher in the presence of 1-OHD
than in the presence of HD. (The head group of 1-OHD
has the formula C3H7O3, which can be considered half a
glucose head group.)
The effects of glucose and maltose on the rate
constants are not significant because these sugars do not
bind to the vesicular interface. Despite the fact that trehalose
is known to bind to the vesicular surface18,19 in the
concentration range used in this study, the kinetic response
for this additive is very minor. However, all kves values found
for Treh/DHAB solutions were faster than those for pure
DHAB, which can be taken as a further indication of
replacement of water molecules from the vesicular
surface.20–23
Vesicles containing DD show a larger effect on the
decarboxylation reaction than vesicles with HD. This
could be due to tail matching/mismatching.12 Tails of
additives that match in length with the tails of the double-
tailed amphiphiles have less impact on the packing of the
vesicular bilayer than in the case of mismatching tails,
therefore mismatching amphiphile tails are expected to
have a stronger negative effect on the vesicular rate
constant than in the case of matching tails.
An important bio-relevant additive in class B is
cholesterol. At low concentrations cholesterol induces
homophospholipid association in fluid bilayers, which is a
direct result of the hydrophobic effect.24 Cholesterol is
said to create organization in lipid bilayers.25–27 Upon
inclusion of cholesterol in the membrane the interamphi-
phile interactions decrease and this increases the hydration
of the interface.28 Cholesterol has the largest hydrophobic
surface area of all additives used in this study. This leads to
a deeper penetration of cholesterol into the bilayer than is
the case for the other additives. As a result, the packing of
the vesicle is more strongly influenced by cholesterol and
Differential scanning
calorimetry measurements
In order to obtain more details about the aggregates
formed from amphiphiles and the additives, differential
scanning microcalorimetric experiments were performed
to measure the main phase transition temperatures (Tm) as
a function of the composition of the bilayers (Fig. 2). The
trends in the Tm of vesicles formed from DHAB and
additives used in this study are similar to those found for
mixed vesicles formed from dimethyl-di-n-octadecylam-
monium chloride and organic additives.29
However, upon addition of small amounts (<30mol.%)
of all additives a slight decrease in Tm was found. Above
30mol.%, C12Mal and C12Glu both give a further decrease
in the main phase transition temperature upon increasing
the mole fraction of additive. However, cholesterol, DD
and HD show an increase in Tm at high mole fractions of
additive. The higher the melting point of the pure additive,
the higher Tm becomes. The majority of the additives
induce phase transitions that occur below 358C, therefore
the chemical behavior of the mixed vesicles can be
compared to one another at this temperature because the
vesicles are all in the liquid crystalline state. Unfortunately
the data do not allow a definite conclusion regarding a
correlation between the effects of the additives on the
catalysis (kves) of the 6-NBIC decarboxylation and on the
phase transition temperature of the tails (Tm).
Activation parameters
For a further characterization of the catalytic effects
induced by mixed vesicles the isobaric activation
parameters were measured for a selection of vesicles
containing 50 mol.% of the additives. The enthalpies and
entropies of activation were calculated using:30
ꢀ
ꢁ
z
R
z
RT
kves
h
D Sꢇ D Hꢇ
ln
¼
ꢂ
(3)
kBT
leads to the largest decrease in kves
.
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254
M. G. M. JONGEJAN, J. E. KLIJN AND J. B. F. N. ENGBERTS
-31
-32
-33
-34
-35
-36
-37
-38
-39
-40
-41
-42
The entropy of activation only provides a minor
contribution to the Gibbs energy of activation, however
it does compensate in part for the enthalpy of activation.
As was seen from the DSC data, vesicles containing DD
have a Tm that lies above 358C at 50 mol.%. Although the
values given for DzGꢇ (and the other activation
parameters) were calculated at 358C, these data were
obtained using the slope for the temperatures above Tm.
The correlation between these data points is good enough
to make the calculated activation parameters of the
different DHAB/additive systems comparable; DD has a
negative TDzSꢇ contribution, C12Glu is associated with a
slightly positive value for TDzSꢇ and DHAB and
trehalose have approximately the same entropic con-
tribution. Thus the vesicular solutions that have the
largest kves values also have the largest values calculated
for the enthalpy of activation and the largest positive
TDzSꢇ contribution.
0.0030 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036
1/T (K-1)
Figure 3. Eyring plot for the decarboxylation of 6-NBIC in
DHAB vesicles containing 50 mol.% DD (&), 50 mol.%
C12Glu (*), 100 mol.% DHAB (þ) and 50 mol.% Treh (ꢆ)
in which kB is the Boltzmann constant, h is the Planck
constant, T is the absolute temperature, R is the gas
constant, DzSꢇ is the entropy of activation and DzHꢇ is the
enthalpy of activation.
Cryo-transmission electron microscopy
Several cryo-TEM pictures were taken of mixed DHAB–
C12Mal vesicles because single-tailed surfactants can
solubilize vesicles into mixed micelles. The cryo-TEM
pictures of 5 mM total concentration of DHAB vesicles
containing 0, 10 and 45 mol.% C12Mal were taken at
358C. As can be seen from the kinetic data, the maximum
observed rate constant for the decarboxylation reaction is
observed at ca. 10 mol.% C12Mal (Fig. 1C). In the absence
of C12Mal (Fig. 5A) and in the presence of 10 mol.%
C12Mal (Fig. 5B) the vesicles are spherical. No micelles
are observed. However, at 45 mol.% C12Mal, holes and
discontinuations in the bilayer can be seen (Fig. 5C). This
will affect the overall hydration of the interface and
therefore influence the decarboxylation reaction of 6-
NBIC, consistent with the observed kinetic data. As long
as the vesicles are still spherical, the reaction is
accelerated by the presence of C12Mal. When the bilayer
is disturbed (structures containing more defects) the
vesicular rate constant decreases. Upon a further increase
of the C12Mal content the bilayer is even further disturbed
and hence the vesicular rate constant decreases even
more.
Eyring plots (Fig. 3) show that there is a break in the
slopes around the main phase transition temperature. The
data points above Tm are of good quality and therefore
these slopes were used for calculations of the activation
parameters. Below the Tm the turbidity of the vesicular
solutions increases and the stability of the samples
decreases. Hence, the quality of the raw kinetic data is
less satisfactory, leading to more scattering of the data
points. This is a common feature of vesicular catalysis.5
In Fig. 4 the calculated activation parameters at 358C
are shown, because this is the temperature at which most
of the kinetic experiments were performed. In agreement
with the literature, the main contribution to the Gibbs
energy of activation comes from the enthalpy.31 The
errors in the calculated values for TDzSꢇ are rather large.
SUMMARY
In this study a kinetic analysis of the decarboxylation of
6-NBIC in DHAB vesicles in the presence of sugars,
long-tailed sugars, cholesterol and alcohol additives has
been made. One of the additives to DHAB vesicles leads
to an increase in the vesicular rate constant relative to
DHAB vesicles, and three of the additives have no
significant effect on the rate constants. All other additives
studied lead to a decrease in the vesicular rate constant.
The natural logarithm of the vesicular rate constants
Figure 4. Activation parameters for DzHAB vesicles contain-
ing 50 mol.% of different additives: D Gꢇ (lined bar), DzHꢇ
(open bar) and TDzSꢇ (filled bar)
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DECARBOXYLATION OF 6-NITROBENZISOXAZOLE-3-CARBOXYLATE
255
Figure 5. Cryo-TEM pictures: (A) 100 mol.% DHAB in 2.25 mM NaOH at 358C; (B) 10 mol.% C12Mal; (C) 45 mol.% C12Mal. The
bars represent 100 nm
measured varies linearly with the mole fraction of
additive for most additives. The monohydric single-tailed
alcohols and cholesterol showed the largest decrease of
in water concentration) of the vesicular interface, thereby
slowing down the vesicular rate constant. In the case of
the n-alkyl pyranosides a balance between the positive
effect of the water displacement and the negative effect of
the membrane disruption caused by the tails seems to
determine the magnitude of the vesicular rate constant.
The hydroxyl groups of the glycerol ether probably also
have the capability to displace water but because there are
fewer hydroxyl groups at the interface the positive effect
is less pronounced.
The DSC measurements indicate that most DHAB/
additive compositions have a Tm below the temperature at
which the kinetic measurements were carried out,
therefore the kinetic data can be compared because all
systems are in the liquid-crystalline state (at <30 mol.%
additive). The Eyring plots show that the enthalpy
of activation is the largest contributor to DzGꢇ. The
additives with the highest values for kves (pure DHAB and
trehalose) also had the largest positive value for TDzSꢇ.
k
ves. The glycerol ethers and the sugar-based surfactants
showed behavior in-between that of the monohydric
single-tailed alcohols and the sugars, which seemed to
have practically no effect on the rate constant. The sugar-
based surfactants induced larger vesicular rate constants
than the glycerol ethers.
In the case of C12Mal the relationship between kves and
the mole fraction of additive was not linear. Cryo-TEM
showed that when (partial) solubilization does not
yet play a role for mixed vesicles containing DHAB
and C12Mal the rate constant is increased relative to
the vesicular solution containing only DHAB. Once the
C12Mal starts inducing defects in the vesicles, the rate
constant becomes smaller than that in pure DHAB
solutions.
A likely explanation for the observed increase in the
vesicular rate constant of vesicles containing C12Mal and
C12Glu compared to vesicles containing DD is that water
is being displaced from the interface by the sugar
hydroxyl groups. This effect is known from the
literature.15,16 The hydrophobic tails of DD, C12Mal
and C12Glu have the ability to change the overall packing
of the aggregate and possibly alter the hydration (increase
Acknowledgments
Dr M. C. A. Stuart is gratefully acknowledged for taking
the cryo-TEM pictures. The NRSC-C is acknowledged
for their financial contribution.
Copyright # 2006 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2006; 19: 249–256

256
M. G. M. JONGEJAN, J. E. KLIJN AND J. B. F. N. ENGBERTS
16. Griffiths PC, Pettersson E, Stilbs P, Cheung AYF, Howe AM, Pitt
AR. Langmuir 2001; 17: 7178–7181.
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