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DUTTA AND TIWARI
reported high enantioselectivity for alkylation of
N-(diphenylmethylene)glycine-tert-butyl ester in lipo-
somes. Recent studies on use of liposomes as reaction
media for 1,3-dipolar cycloaddtion reactions[18] and
Michael addition reaction[19] have also highlighted the
critical role of liposomal interface in accelerating the
rates of these reactions. Kinetic and mechanistic investi-
gations of chemical processes in liposomal assemblies
have been very few in number. For example, several
modes of liposomal reactivity under identical conditions
inside the liposome or at the liposome-water interface. It
may be possible that the reaction occurs at all three
“locations” simultaneously with either one or two of
them dominating the reaction outcome—depending on
the conditions employed. This situation also complicates
the quantitative discussion of kinetic data—especially for
comparison of rates in bulk media with those occurring
at the interface. In order to overcome this limitation, the
rate constants in the present report have been designated
as “apparent” rate constants, although all experimental
kinetic data could be best modeled as first order
(or pseudo first order) rate processes.
Another important consideration would be the pH of
the solution, which can be influenced by the presence of
a weak base (morpholine, pKa = 8.4). For the concentra-
tions of morpholine used in this study, the pH of the
aqueous medium was found to be greater than 9 and var-
ied slightly for different concentrations of morpholine
used. No buffer solution was used to control the
pH of the reaction medium. The possibility of
competitive reactions with hydroxide ions at such high
pH cannot be completely ruled out. However, the
stoichiometric conversion of the substrates to the product
N-(2,4-dinitrophenyl)morpholine indicates that any com-
petitive reactions may be negligible in extent.
were investigated by Menger and Azob[20] as
a
cytomimetic model. The observed rates were justified on
the basis of hydrophilic nature of the functional groups
present. Further investigation revealed the critical role of
interliposomal transfer of reactant molecules during
collisions between liposomes.[21]
An additional incentive for studying chemical pro-
cesses in liposomes is that it provides an opportunity to
model phospholipid membranes in vitro while overcom-
ing many of the experimental limitations associated with
in vivo studies. These membranes are fundamental
building blocks for cellular structures and are capable of
controlling the outcome of numerous biological
processes.[22–25] In recent years, this approach has been
explored extensively to design protocells that can mimic
cellular complexity in the form of cascading enzymatic
reactions,[26] synthesis of proteins[27] and the effect of
compartmentalization over peptide synthesis.[28,29]
The present work explores the model SNAr reaction
of 1-halo-2,4-dinitrobenzene (DNXB) with morpholine
in liposomal assemblies composed of 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-
sn-glycero-3-phosphocholine (DMPC). Various factors
that could influence the reaction outcome such as loca-
tion of the substrate vis-à-vis the liposomal assembly,
composition of the liposomes, and concentration of lipid
constituting the liposomes were examined. Varying the
halide substituent affected the sensitivity of the substrate
to the liposomal reaction medium and thus influenced
the rate of the reaction. The results are discussed in terms
of the hydrophobicity and leaving group ability of the
substrate.
3 | KINETICS IN LIPOSOME
VERSUS AQUEOUS MEDIA
The primary mechanistic concern about any reaction car-
ried out in liposomes would be to determine which loca-
tion/s is/are most conducive for the reaction to occur. In
order to determine whether the model SNAr reaction pri-
marily occurred in the aqueous phase, inside the lipo-
some or at the interface, the rate of product formation
was determined under three different reaction condi-
tions. Initially, the rates of nucleophilic substitution reac-
tion between 1-fluoro-2, 4-dinitrobenzene (DNFB) and
morpholine were determined in the presence of
liposomes.
Two different approaches were adopted to measure
the kinetics of the reaction in liposomal media. In the
first approach, the DNFB substrate was added to liposo-
mal dry film before formation of liposomes and thus, got
“encapsulated” inside the liposome due to its low solubil-
ity in water. In the second case, the substrate was allowed
to get adsorbed on the liposome interface after the forma-
tion of liposome from the dry film; that is, it was
“incubated” to yield the liposomal solution of DNFB.
Given the low solubility of substrate and low membrane
permeability of pure DPPC liposomes and our kinetic
data, it can be safely assumed that majority of the
2 | RESULTS AND DISCUSSION
Unlike conventional kinetic studies in homogeneous
reaction media, the kinetic studies for micro-
heterogeneous media are complicated by the fact that the
reaction dynamics may depend on the exact location of
the reaction—in the bulk phase or at the interface. For
example, reactions taking place in liposomal media may
occur at any one of the three “locations”—in bulk water,