A R T I C L E S
Kumar and Oliver
solution techniques such as chromatography for product char-
acterization and kinetic analysis of reactions in monolayer
films.13 To our knowledge, there is no report where a technique
capable of structure determination has been utilized to unam-
biguously identify reaction products and provide quantitative
measurements of reaction yield and kinetics at the air-water
interface.
The components of a reaction designed to form a discrete
product at the air-water interface are shown in Figure 1. A
monolayer composed of 1 and 2 is formed on an aqueous
subphase. The thioester 1 is the single reactive electrophile, and
the amino group of 2 is the only nucleophile present in the
monolayer. The condensation products of this reaction, dipeptide
4 and thiol 3, remain at the interface after the reaction. The
N-terminus of 1 is derivatized, precluding further reaction.
In our initial report on this reaction, the monolayer film was
isolated from the air-water interface, and the reaction products,
including dipeptide 4, were characterized by thin-layer chro-
matography, mass spectrometry, FT-IR, and 1H NMR spectros-
copy.11
Figure 1. Reaction scheme of derivatized amino acids designed to give a
discrete product at the air-water interface.
molarities of reactive groups.8 Proximity effects from concen-
trating reactive functionalities in a two-dimensional plane can
result in rate accelerations of reactions over similar conditions
in homogeneous solution.9 In addition, imposing structural order
over the monolayer film reduces the rotational and translational
entropies of the surface molecules, giving rise to reaction
pathways that can be different from pathways in solution.10
However, the mechanisms that underlie reactivity in monolayers
are not well understood.
As a model for the amide bond-forming reaction catalyzed
by the ribosome, we investigated the behavior of a related
system in a monolayer film. We have previously shown that a
long chain thioester of an amino acid spontaneously undergoes
an acyl transfer reaction with an amphiphilic nucleophile when
the two reactants are held in proximity in a monolayer (see
Figure 1).11 To quantify the rate acceleration provided by
proximity effects within the monolayer film, we now report the
kinetics of this amide bond-forming reaction at the air-water
interface.
The kinetics of reaction of several surface bound amphiphiles
has been investigated. However, the majority of published
studies monitor reactions at the air-water interface using surface
techniques such as changes in surface area, surface pressure,
surface potential, viscosity, or surface spectroscopy.12 These
methods are inadequate for structural characterization of reaction
products, and the product/reactant ratios can only be obtained
indirectly. Only a small number of investigations have employed
1
We have developed protocols that allow us to use H NMR
spectroscopy to evaluate product formation of single compres-
sion experiments on a Langmuir-Blodgett film trough. This
allows accurate measurement of reaction progress at each time
point analyzed. Herein, we describe the effect of pH, surface
pressure, and temperature on the kinetics of an amide bond-
forming reaction at the air-water interface.
Experimental Section
General. The synthesis and characterization of 1, 2, and 4 have been
described earlier.11 CDCl3 used to collect surface products was filtered
through NaHCO3 and Na2SO4 immediately prior to use. All chemicals
used in buffering the subphase were molecular biology grade obtained
from Sigma. Water was purified with a Millipore Nanopure system to
a resistivity of at least 18 MΩ cm.
Hydrophobic Derivatization of Glass Slides. Clean microscope
slides were subjected to the RCA cleaning method.14 The slides were
first immersed in a boiling solution of H2O/H2O2/NH4OH (5:1:1) for
10 min and then in a boiling solution of H2O/H2O2/HCl (6:1:1) for 10
min. The slides were rinsed with water and then dried overnight in an
oven at 180 °C. The clean slides were derivatized by immersion in a
solution of octadecyltrichlorosilane in hexadecane (2%) for 2 h.15 The
slides were rinsed with CHCl3 and dried under a stream of N2. The
derivatized slides were stored in a dark desiccator prior to use.
Pressure-Area Isotherms. Pressure-area isotherms were obtained
with a film balance equipped with a Wilhelmy platinum plate connected
to a microelectronic feedback system for surface pressure control. The
film trough was placed in a specially constructed polycarbonate cabinet
to minimize dust exposure and to control the humidity. The temperature
of the subphase was maintained by circulating water through the walls
of the trough. The subphase pH was maintained by a buffered solution
(10 mM NaOAc, 1 mM EDTA, 0.5 M NaCl). Amphiphiles were spread
with a 50 µL syringe from ∼1 mg/mL solutions in CHCl3 by applying
the solution to at least 10 different places on the water surface. For
mixed monolayers, a chloroform solution containing the two am-
phiphiles was spread. Solvent was allowed to evaporate for 30 min.
The monolayer was compressed at a speed of 3 × 1017 Å2/min.
Equilibrium Spreading Pressures. Equilibrium spreading pressures
were measured by depositing 300 µL of ∼1 mg/mL solutions of the
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