Pseudo-Interphase of Liposome Promotes 1,3-Dipolar Cycloaddition Reaction of Benzonitrile Oxide and N -Ethylmaleimide in Aqueous Solution

Article abstract of DOI:10.1021/acs.jpcb.5b03762

The hydrophobic interior of a liposome membrane was used as a platform for the organic synthesis of hydrophobic compounds in water. The 1,3-dipolar cycloaddition of benzonitrile oxide (BNO) and N-ethylmaleimide (EMI) in liposome suspensions was carried ou

Full text of DOI:10.1021/acs.jpcb.5b03762

Article
pubs.acs.org/JPCB
Pseudo-Interphase of Liposome Promotes 1,3-Dipolar Cycloaddition
Reaction of Benzonitrile Oxide and N‑Ethylmaleimide in Aqueous
Solution
Fumihiko Iwasaki, Keishi Suga, and Hiroshi Umakoshi*
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka,
Osaka 560-8531, Japan
S
* Supporting Information
ABSTRACT: The hydrophobic interior of a liposome
membrane was used as a platform for the organic synthesis
of hydrophobic compounds in water. The 1,3-dipolar cyclo-
addition of benzonitrile oxide (BNO) and N-ethylmaleimide
(EMI) in liposome suspensions was carried out, and an
increase in the reaction rate constant was observed depending
on the liposome characteristics. While the reaction rate
constant in 1,4-dioxane was 1.5 times higher than that in
water, the reaction rate constant in an aqueous solution of
cationic 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP) liposome was 3 times higher than in water. The
amount of substrate, BNO, accumulated in the DOTAP liposome was higher than that in 1,2-dipalmitoyl-3-trimethylammonium-
propane (DPTAP), indicating that BNO prefers to be distributed in the liposome membrane in the liquid-disordered phase. The
membrane polarity, GP340, as monitored by Laurdan, varied with the presence of BNO, while EMI slightly affected the membrane
properties of the liposomes. These results suggest that the pseudo-interphase afforded by the liposome membrane can promote
the 1,3-dipolar cycloaddition between BNO and EMI in water.
Diels−Alder reactions.18−21 Micellar suspensions can provide a
INTRODUCTION
■
hydrophilic−hydrophobic interphase between the bulk solution
Water is an ideal solvent, as it is harmless, nonflammable, and
and hydrophobic interior of the assembly. It has been reported
stable. Environmentally friendly chemical processes with high
that the pseudo-phase model22 can be applied to the aldol
atomic efficiency1 have attracted significant attention, and thus,
numerous organic reactions in water have been developed,2
even though the polarity of water molecules and the insolubility
of reactants usually prevent reactions from occurring in the
aqueous phase. To overcome such disadvantages, several kinds
of catalysts that work at the “interphase” have been reported.3−5
For example, phase-transfer catalysts (PTC) have been
developed as a novel method to catalyze the transfer of
reactants across one liquid phase to another immiscible
phase.6−9 The reactants can accumulate in the interphase
between two liquid media, where the interphase region can act
as a reaction site. The advantage of PTC can be seen in the
enantioselectivity of products; nevertheless, they require polar
organic solvents and a strong base (or acid) and are not
regarded as environment friendly. Organocatalysts, such as L-
proline and its derivatives,10−13 have attracted significant
attention from the viewpoint of metal-free and organic
solvent-free reaction systems. Several organic syntheses have
been reported in water using organocatalysts,14−18 including
Diels−Alder, aldol, and Michael reactions.
reaction and Diels−Alder reaction in micellar or emulsion
systems.18,19 Meanwhile, 1,3-dipolar cycloadditions, which are
similar to the Diels−Alder reaction, can be influenced by the
degree of solvation as well as the frontier molecular orbitals
(FMOs) of the reactants.23,24 In homogeneous solution
systems, it is assumed that the relative dielectric constant (ε)
and proticity of the reaction media play important roles on the
reaction. Although the reaction mechanism might be different
in micelle solution, the properties of the hydrophilic−
hydrophobic environment around the reactants may also
regulate the reactions.
Liposomes, which consist of a lipid bilayer with a 5 nm thick
hydrophobic interior, also provide a hydrophobic−hydrophilic
interface. Such hydrophobic interiors can be utilized as a
nanoreactor. In our previous works, (bio)chemical conversions
have been carried out at the surface of liposome mem-
branes.25−29 Herein, the pseudo-interphase, defined as the
surface region of liposome membranes where the hydrophilic
aqueous phase and hydrophobic liposome interior are enclosed
Recently, highly organized interfaces, such as the surfaces of
self-assembled amphiphilic molecules, have been utilized to
achieve successful chemical conversions in water. For example,
some micellar aggregates have been reported to promote
Received: April 20, 2015
Revised: July 3, 2015
Published: July 6, 2015
© 2015 American Chemical Society
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within a nanometer scale, is expected to act as a platform for
reactant localization. In addition, the surfaces of the liposome
membranes are estimated to be hydrophobic similar to some
organic solvents.30 It is therefore indicated that the reactants
localized at the liposome surface could be affected by the
surrounding environment, i.e., membrane polarity, etc. Lip-
osome suspensions could be suitable systems for metal-free and
organic solvent-free organic syntheses, because the character-
istics of the microscopic environment at liposome surfaces
(fluidity and polarity among others) are designable; however,
previously reported works have not focused on the character-
ization and design of self-assemblies.
BNO solution was obtained. The formation of BNO was
determined by UV−vis spectroscopy (UV-1800; Shimadzu,
Kyoto, Japan) and a high resolution double-focusing magnetic
sector mass spectrometer (JMS700; JEOL, Tokyo, Japan),
where the conversion of BO was >85%. The prepared BNO
solution was stable for at least 3 h, and the experiments were
performed soon after the BNO was prepared.
Preparation of Liposomes. A chloroform solution with
lipids was dried in a round-bottom flask by evaporation under a
vacuum.35 The obtained lipid thin film was dissolved in
chloroform again, and the solvent was evaporated. The lipid
thin film was kept under a high vacuum for at least 3 h, and was
then hydrated with distilled water at room temperature. The
liposome suspension was frozen at −80 °C and thawed at 50
°C to enhance the transformation of small vesicles into larger
multilamellar vesicles (MLVs). This freeze−thaw cycles were
performed five times. MLVs were used to prepare the LUVs by
extruding the MLV suspension 11 times through two layers of
polycarbonate membranes with mean pore diameters of 100
nm using an extruding device (Liposofast; Avestin Inc., Ottawa,
Canada).
In this study, the 1,3-dipolar cycloaddition20,31−34 between
benzonitrile oxide (BNO) and N-ethylmaleimide (EMI) was
carried out in the presence of various kinds of liposomes. This
is the first report which details the effects of liposome
membranes on this reaction. Because the surface of liposome
membranes is hydrophilic environment (water rich), the
accumulation of reactants at the membrane surface could be
regarded as a solvation effect in the aqueous phase. Here, three
types of aqueous solutions were used as reaction media: (i) 1,4-
dioxane/water binary mixtures (0−100 v/v% 1,4-dioxane), (ii)
micellar solutions (cetyltrimethylammonium bromide (CTAB),
sodium dodecylsulfate (SDS)), and (iii) liposome suspension
systems. The interphase region between hydrophobic medium
and hydrophilic medium was defined a pseudo-interphase, and
its effects on the 1,3-dipolar cycloaddition are discussed. The
microscopic environment of each liposome was characterized
using fluorescence probes;35 membrane polarity was analyzed
by 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) and 6-(p-
toluidino)-2-naphthalenesulfonic acid sodium salt (TNS). As
an indicator of water solvation in each medium, the relative
dielectric constants (ε′) of the liposome suspensions were
estimated. The kinetics of the 1,3-dipolar cycloaddition
between BNO and EMI were analyzed by pseudo-first-order
kinetics. The distribution of each reactant in the liposome
membrane was evaluated by ultrafiltration, and the relationship
between the adsorption of reactants on the liposome and the
dehydration of the membrane surface were then considered.
Finally, the reaction kinetics at the pseudo-interphase are
discussed, where the possible role of the liposome is to
accumulate the reactants and to regulate the reaction in
aqueous media.
Evaluation of Membrane Polarities. Laurdan is sensitive
to the polarity around the molecule itself, and its fluorescence
properties enable to evaluate the surface polarity of lipid
membranes.30 The Laurdan emission spectra exhibit a red shift
caused by dielectric relaxation. The emission spectra were
measured with an excitation wavelength of 340 nm, and the
general polarization (GP340), the membrane polarity, was
calculated as follows:
GP = (I440 − I490)/(I440 + I490
)
340
where I440 and I490 represent the fluorescence intensity of
Laurdan at 440 and 490 nm, respectively. To compare the polar
environment of the liposome or micelle surface and solvent, the
apparent polarity of liposome, ε′, was calculated (see
Supporting Information Figure S1). The total concentrations
of lipid and Laurdan were 100 and 1 μM, respectively.
For measuring membrane polarity by TNS, a TNS/ethanol
solution (100 μM) was used instead of a Laurdan/ethanol
solution and the rest of the process is the same as that for the
measurement using Laurdan.
1,3-Dipolar Cycloaddition of BNO and EMI (Scheme
1). 10 μL of BNO solution (25 mM in 1-propanol) were mixed
with 1 mL of sample solution, composed of a dioxane/water
mixture, liposome suspension (lipid concentration = 0.25 mM),
or micelle solution (lipid conc. = 250 μM). The sample
solution was incubated for 30 min at room temperature, and
then, 5 μL of EMI solution (200 mM in 1-propanol) were
added to initiate the 1,3-dipolar cycloaddition. The total
concentrations of BNO and EMI were 250 μM and 1 mM,
respectively.
Kinetics Measurement of UV Absorbance. The UV
absorbance spectra of sample solution was analyzed from 500
to 200 nm, using a UV spectrophotometer (UV-1800;
Shimadzu, Kyoto, Japan). The UV absorbance at 278 nm is
originated from product20,31 that is distinguishable from the
peak of BNO (258 nm) or EMI (300 nm). The reaction was
started in a quartz cuvette, and a time course of the UV
absorbance at 278 nm was measured for 15 min at room
temperature. The reaction rate constant was analyzed via
pseudo-first-order kinetics, because of the excess of EMI (4-
fold), which is calculated as follows:
EXPERIMENTAL METHODS
■
Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-
choline (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC), 1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP), and 1,2-dipalmitoyl-3-trimethylammonium-propane
(DPTAP) were purchased from Avanti Polar Lipid (Alabaster,
AL, USA). 6-Lauroyl-2-dimethylaminonaphthalene (Laurdan),
and 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt
(TNS) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Benzaldehyde oxime (BO) and sodium hypochlorite
solution (NaClO(aq)) were purchased from Kanto Chemical
(Tokyo, Japan). Other chemicals were purchased from Wako
Pure Chemical (Osaka, Japan). These chemicals were used
without further purification.
Benzonitrile oxide (BNO) was prepared according to the
literature.20 In brief, 21.6 μL of BO were dissolved in 2 mL of
the NaClO(aq)/1-propanol solution (50:50). 0.36 g of NaCl
was added to this solution to separate it into two phases. After
the upper phase was taken and diluted by 1-propanol, 25 mM
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indicating that they were in the liquid-disordered phases.29 In
contrast, DSPC and DPTAP showed higher GP340 values at
room temperature. Because the phase transition temperatures
of DSPC and DPTAP are 55 and 45 °C, respectively, DSPC
and DPTAP were in the solid-ordered phases. Because Laurdan
peak shifts are dependent on environmental hydrophobicity,
GP340 values can indicate the hydrophobicity as well as the
relative dielectric constants. To investigate the relative dielectric
constant, the nonpolarity factor (NF340) was examined; NF340
values reveal the degree of nonpolarity of the solvents and were
applied to characterize the liposomes. The NF340 values were
calculated using the ratio of the fluorescence intensities of
Laurdan in organic solvents and water (see Experimental
Methods and Figure S1). The relative dielectric constant, ε′,
can be calculated from NF340 values, suggesting that the
approximate amount of water at the liposome membrane
surface can be estimated by analyzing the GP340 values (Figure
1). Liposomes in the liquid-disordered phases showed high ε′
Scheme 1. 1,3-Dipolar Cycloaddition of Benzonitrile Oxide
(BNO) and N-Ethylmaleimide (EMI)
a
a
(1) BO, (2) BNO, (3) EMI, (4) product.
⎛
⎝
⎞
⎠
A278
A278,e
⎜
⎟
⎟
−ln 1 −
= kt
⎜
where A278 and A278,e represent the UV absorbance at 278 nm at
any moment t in the reaction and that at 15 min (end of the
reaction), respectively. k is the reaction rate constant, and t is
the time. The concentrations of reactants were 0.25 mM for
BNO and 1.0 mM for EMI. The concentration of lipid was 0.25
mM. The relative reaction constant, krel, was calculated as
follows:
krel = k/kwater
where kwater represents the reaction rate constant in water.
Adsorption of Reactants onto Liposome. The adsorp-
tion amounts of the reactants were also evaluated by UV
spectrophotometer. 10 μL of BNO solution (25 mM in 1-
propanol) were mixed with 1 mL of liposome suspension (lipid
concentration = 0.25 mM) and incubated for 30 min at room
temperature to measure the adsorption of BNO. To measure
the adsorption of EMI, 5 μL of N-ethylmaleimide solution (200
mM in 1-propanol) were mixed with 1 mL of liposome
suspension (lipid concentration = 0.25 mM) and incubated for
30 min at room temperature. After the incubation, the liposome
and reactants adsorbed on the membrane were removed with
the ultrafiltration unit USY-20 (molecular weight cutoff:
200 000, Advantec Toyo, Ltd., Tokyo, Japan). The adsorption
percentages were calculated from the difference in UV
absorbance of the solution before and after the filtration:
Figure 1. Characteristics of liposome. Membrane polarity (GP340) was
measured from the experiment using Laurdan, and relative dielectric
constant (ε′) was calculated by the fitting equation from Figure S1.
values (ε′ = 20−25), while low ε′ values (ε′ = 3−5) were
observed in liposomes in the solid-ordered phases. CTAB
micelles also showed a higher ε′ value, implying that the
membrane surface of CTAB micelles is similar to that of
liposomes in liquid-disordered phases. It is known that the
permeability of small molecules across liposome membranes
can be influenced by the phase states of the liposomes.37
Furthermore, it has been reported that the distribution of BNO
in positively charged DOTAP liposomes is higher than that in
zwitterionic DOPC liposomes.38 It is therefore expected that
the reactants can accumulate at the surface of liposomes, and
the reaction can occur at the pseudo-interphase of the
liposomes.
2. Effect of Liposomes on 1,3-Dipolar Cycloaddition.
In the 1,3-dipolar cycloaddition of BNO and EMI, the
surrounding dielectric environment is a key factor.34 Notably,
the reaction between BNO and EMI was carried out in water
(Figure S2). In order to estimate the effects of the reaction
media on the 1,3-dipolar cycloaddition between BNO and EMI,
the relative dielectric constant ε′ and the relative reaction
constant, krel, were plotted (Figure 2). With the 1,4-dioxane/
water systems, the krel values became higher with a decrease in
the relative dielectric constant of the solvent. The relative
reaction rate constant in 1,4-dioxane (ε = 2.2) was 1.6 times
higher than that in water (ε = 78), indicating that the
Adsorption percentage
= ((Ainitial − Afiltrated)/Ainitial) × 100
where Ainitial and Afiltrated represent the absorbance of the
reactant (BNO or EMI) in the solution before and after
ultrafiltration, respectively.
RESULTS AND DISCUSSION
■
1. Characterization of Liposomes. Pseudo-interphase
reactions constitute an important group in chemical reactions
in self-assembled systems.38 Notably, the polarity and proticity
of solvents can regulate the partitioning of reactants; the
microscopic environment of the liposome surface can also
regulate the 1,3-dipolar cycloaddition reaction, as evaluated in
this study.20 In order to investigate the membrane properties of
the liposomes, the general polarization (GP340) was estimated
using Laurdan. The GP340 values varied with the acyl chain
length and surface charge of the lipid molecules that comprised
the liposome membranes (Table S1). POPC and DOTAP
liposomes showed lower GP340 values (GP340 < −0.2),
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was more promoted in liposomes in the liquid-disordered
phases (ε′ = 20−25) as compared to that in liposomes in the
solid-ordered phases. Among the liposomes used in the present
study, DOTAP liposome showed the highest promotion effect
on the reaction, indicating that the DOTAP liposome could
provide a better environment for the reaction. Rispens and co-
workers reported that 1-propanol containing 40 M of water was
the best medium for the 1,3-dipolar cycloaddition between
BNO and EMI.20 Although the surface charge densities of
liposomes or micelles differed (Table S1), no relationship was
obtained between the surface charge density and the promotion
of the reaction. It is notable that the krel values were not so high
just after the mixing of the reactants in each medium (Figure
S3). To find a better environment and to promote this reaction
in liposome systems, the condensation of the reactants should
be considered.
3. Accumulation of BNO and EMI in Liposome
Membranes. The local concentrations of the reactants were
estimated based on the ultrafiltration method.38 The accumu-
lation of the reactants on the liposome membranes could be
critical to the enhancement of the 1,3-dipolar cycloaddition
reaction between BNO and EMI at the DOTAP liposome
membrane. Figure 3 shows the amount of BNO and EMI
distributed on each liposome. DOTAP showed a higher
adsorption percentage of BNO as compared with POPC
(Figure S4). DPTAP, the cationic liposome in the solid-ordered
phase, did not significantly promote the distribution of BNO as
compared to DOTAP, while the distribution of BNO on
DPTAP at 60 °C increased to 34%, when DPTAP was in the
liquid-disordered phase. The BNO distribution analysis
indicated that BNO was distributed on the DOTAP liposome
not only because of the positive charge but also because of the
phase state. Only a small amount of BNO was distributed in the
CTAB micelle. In contrast, BNO molecules could accumulate
in the membrane. DOTAP, the cationic liposome in the liquid-
disordered phase, formed a suitable environment for BNO
accumulation. In the case of EMI, the distribution ratio was not
much higher than that of BNO, and the adsorption percentage
values were almost similar within the liposomes. EMI is more
Figure 2. Reaction rate constant in various conditions. The reaction of
BNO (0.25 mM) and EMI (1.0 mM) was carried out in water at 25
°C. The reaction was initiated after 30 min incubation of BNO with
the medium. For the 1,4-dioxane/water system (open circle), 1,4-
dioxane/water was used as the medium to vary the relative dielectric
constant. For liposomes (closed circle), liposome suspensions (0.25
mM) were used as the reaction medium, and 5 mM micelle solutions
were used for micelles (closed diamond). Each point indicates the
following: (1) DSPC, (2) DPTAP, (3) POPG, (4) POPC, (5) POPC/
DOTAP, (6) DOTAP, (7) CTAB, (8) SDS. Rate constant values are
relative values compared to the reaction conducted in water (ε′ = 78).
hydrophobic environment could promote the 1,3-dipolar
cycloaddition. BNO is hydrophobic and thus prefers hydro-
phobic environments.38 Because 1,4-dioxane is an aprotic
solvent, the reaction between BNO and EMI in 1,4-dioxane/
water systems would be dependent on the solvation of the
reactants. With the micellar suspensions, a nonpromotion effect
was observed even though the surface of the micelles was
hydrophobic (ε′ = 35−45). This result indicated that the
reactants could not accumulate on the surface of the micelle
aggregates. In the case of the liposomes, the krel values increased
by 2.2−2.8 times as compared to the water solution, depending
on the characteristics of the liposomes. Specifically, the reaction
Figure 3. Adsorption Behavior of Substrate Molecules on Liposome Membrane. Each reactant (BNO: 0.25 mM and EMI: 1.0 mM) was incubated
with liposome suspension (0.25 mM) in water for 30 min. Adsorption percentage was calculated from the difference of the absorbance before and
after filtration (adsorption percentage = ((Ainitial − Afiltrated)/Ainitial) × 100). The absorbance was measured at 25 °C unless notified. (a) BNO
adsorption, (b) EMI adsorption.
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Figure 4. Change in values of fluorescent probes. (a) Intensity of TNS fluorescence was measured. TNS (1.0 μM), DOTAP liposome (100 μM),
and optional amount of substrates were incubated for 30 min before measurement. (b) Fluorescent spectrum of Laurdan was obtained. Laurdan (1.0
μM), DOTAP liposome (100 μM), and optional amount of substrates were incubated for 30 min before measurement. GP340 values were calculated
as written in Experimental Methods section.
Figure 5. Scheme of the reaction at the interface of liposome membrane.
hydrophilic than BNO: the calculated log P values for BNO and
EMI were 1.82 and −0.18, respectively.39 Although the
distributed amount of EMI was smaller than that of BNO, it
was assumed that EMI preferred to be distributed on the solid-
ordered membranes, such as DSPC and DPTAP. In general,
1,3-dipolar cycloadditions are concerted reactions.40 Thus, both
reactants must accumulate at the pseudo-interphase region
simultaneously if the reaction occurs around the surface of the
liposome membrane. This indicates that strong interactions
between reactants and liposome membranes might inhibit the
reaction. In the case of the micelle solutions, the mismatched
localization of reactants would inhibit the 1,3-dipolar cyclo-
addition.20 It is therefore important to estimate the
colocalization of BNO and EMI at the surface of the liposomes.
4. Estimation of the Localization of Reactants in
Liposome Membranes. The distributions of the reactants in
the DOTAP liposome membrane were investigated. Based on
the hierarchic binding of the fluorescent probes,41 the
localization behaviors of BNO and EMI were analyzed. TNS
and Laurdan exist in the hydrophobic−hydrophilic interface
region. After these fluorescent probes were embedded in the
liposome membranes, BNO and EMI were added to each
sample, and variations in the membrane properties were
evaluated (Figure 4). After BNO was added, the fluorescence
intensity of TNS decreased and the GP340 values increased,
indicating that BNO could be localized in the regions where
TNS and Laurdan were embedded. It was therefore determined
that BNO could be localized at the hydrophobic−hydrophilic
interface of the DOTAP liposome. On the other hand, the
Laurdan signals were not significantly altered in the presence of
EMI. The fluorescence intensity of TNS decreased after
addition of EMI, indicating that EMI could be localized at
the hydrophilic membrane surface region of the liposomes.
From these results, it can be assumed that these two reactants
were enclosed at the surface of the DOTAP liposome and the
1,3-dipolar cycloaddition would occur in the hydrophilic region
of the membrane (not in the hydrophobic interior region). It is
therefore expected that the accumulation of reactants in the
DOTAP liposome can promote Diels−Alder reactions and 1,3-
dipolar cycloadditions.
The polar environment of the liposome membrane was
monitored by analyzing the variations in GP340 values before
and after BNO distribution (Figure S5). The ΔGP340 value of
DOTAP was highest among the liposomes used in this study,
although the change in the GP340 value of DOTAP was not as
significant as compared to the phase transition from the liquid-
disordered phase to the solid-ordered phase.29 BNO could be
embedded in the hydrophobic−hydrophilic interface region
where water molecules bind, and could replace the water
molecules, resulting in the dehydration of the membrane
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surface (i.e., an increase in the GP340 value). Here, a few percent
of water was excluded from the liposome membrane (the
relative dielectric constant ε′ varied to 25.0 from 26.3; see
below). In the presence of BNO, the dehydration degree of
DOTAP liposome was most significant (Table S2). Based on
the above results, the reactants were distributed in the water-
rich environment on the surface of the DOTAP liposome
(pseudo-interphase), and the reaction was promoted due to the
enclosure of the two reactants.
5. Estimation of the Reaction Rate Constants
Occurring at the Liposome Surface. In this study,
pseudo-first-order kinetics were applied to calculate the
reaction rate constant of BNO and EMI in different media.
Since the reaction rate constant of the 1,3-dipolar cycloaddition
could depend on the degree of solvation and FMO of the
reactants,19,20,34,42 the relative dielectric constant, ε′, is helpful
to understand the effects of the reaction media. In a
homogeneous solution of 1,4-dioxane/water, a linear relation-
ship between krel and ε′ was approximately obtained, indicating
that the solvation of the reactants is critical to promote the
reaction. In the case of the micelle solutions, the krel values were
almost the same as those in water. The result shown in Figure 3
reveals that small amounts of BNO (4.5%) and EMI (2.3%)
were distributed on the micelle membrane, where a non-
promoting effect was observed on the surface of micelle
aggregates. In contrast, 2.1-to-2.8-fold higher krel values were
obtained in liposome suspensions. However, the apparent
reaction rate constant can be divided into two types of
reactions: in bulk water and at the pseudo-interphase of the
liposome (Figure 5). In this way, the calculated reaction rate kcal
can be written as
solvation of BNO in aqueous media; since BNO is a
hydrophobic molecule, BNO might not be solvated in the
bulk aqueous phase. However, when BNO is at the pseudo-
interphase, BNO can be stabilized and solvated by water.
Therefore, the solvation of the reactants by water molecules
could be critical in forming the activated state and promoting
the reaction.
From Table 1, it can also be seen that the kL values in
DOTAP and DPTAP liposome systems were smaller than
those in POPC and DSPC liposomes. This result shows that
adsorption of BNO was not directly related to the value of kL.
Rather, it is indicated that the strong electrostatic interaction
between BNO and liposomes can inhibit the reaction. A high
adsorption of BNO might make BNO more solvated by water
molecules, but a high affinity toward the reactants also
influences the reaction, possibly because of the restriction
imposed on the reactive moiety.
CONCLUSIONS
■
The environment of the pseudo-interphase of liposomes, as
evaluated by Laurdan, was found to be an important factor that
can regulate the accumulation of BNO, and its reaction with
EMI was improved in the aqueous medium. Liposomes draw
both reactants toward the pseudo-interphase and enrich the
reactants to promote the reaction. DOTAP liposomes attract
more BNO such that more reactants are in a favorable
environment for the reaction. The accumulation of BNO at the
membrane surface makes DOTAP liposomes unique in regard
to the adsorption of the reactants, but strong interactions might
not be directly related to the promotion of the reaction. It is
assumed that the liposome membrane surface and hydrated
water play essential roles in promoting the 1,3-dipolar
cycloaddition reaction between BNO and EMI at the pseudo-
interphase. It is assumed that hydrated water and “activated”
BNO would be localized at the liposome membrane surface. In
general, by utilizing the liposome membrane as a designable
interface, novel chemical processes can be developed without
organic solvents.
(reaction rate) = k[BNO]total = kW[BNO]W + kL[BNO]L
where kW is the reaction rate constant for the reaction occurring
in the bulk water phase and kL is the reaction rate for the
reaction occurring at the pseudo-interphase of the liposome;
[BNO]W and [BNO]L are the concentration of BNO in bulk
water and at the pseudo-interphase of the liposome,
respectively. The calculated values of (kL/kW) are shown in
Table 1. The reaction rates at the pseudo-interphase of the
ASSOCIATED CONTENT
* Supporting Information
■
S
Table 1. Values of the Reaction Rate with Liposomes and
Micelle
Calculation of relative dielectric constants (ε′); Kinetic data of
the reaction; Additional data of the reaction rate and absorption
behavior. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acs.jpcb.5b03762.
ε′ [−]
krel (=k/kw) [−]
BNO ads. [%]
kL/kw [−]
POPC
DSPC
19.7
2.69
26.3
4.52
38.5
2.24
2.15
2.75
2.14
0.96
13.4
12.4
38.0
16.7
4.5
10.25
10.27
5.61
DOTAP
DPTAP
CTAB
AUTHOR INFORMATION
Corresponding Author
*Telephone: +81-6-6850-6287. Fax: +81-6-6850-6286. E-mail:
b-ice@cheng.es.osaka-u.ac.jp.
■
7.83
0.11
liposome were much higher than those in water or micellar
solutions. The increase in reaction rate in liposome systems
might be because of hydrated water at the liposome surface.
Because EMI is localized at the surface of the liposome due to
its hydrophilicity, the reaction was considered to take place at
the surface of the liposome, where hydrated water molecules
exist. It has been reported that an increase in the amount of
water also increases the reaction rate;42 therefore, hydrated
water molecules at the liposome surface can play an important
role in the 1,3-dipolar cycloaddition. On the other hand, the
reaction rates decreased in 1,4-dioxane/water systems with
increasing amounts of water. This is possibly because of the
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Funding Program for Next
Generation World-Leading Researchers of the Council for
Science and Technology Policy (CSTP) (GR066), JSPS Grant-
in-Aid for Scientific Research A (26249116), and JSPS Grant-
in-Aid for Research Activity Start-up (25889039). One of the
authors (K.S.) also expresses his gratitude for the Japan Society
for the Promotion of Science (JSPS) and GCOE scholarships.
9777
DOI: 10.1021/acs.jpcb.5b03762
J. Phys. Chem. B 2015, 119, 9772−9779

The Journal of Physical Chemistry B
Article
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