A self-assembled nanofiber catalyst for ester hydrolysis

Article abstract of DOI:10.1021/ja075044n

Catalytic particles based on supramolecular systems have demonstrated significant improvement in the field of catalysis. In this work, histidine-functionalized self-assembling peptide amphiphiles (PAs) were synthesized in order to form self-assembly high-aspect-ratio nanofibers with internal order that can present imidazolyl groups capable of ester hydrolysis. Self-assembly of the molecules making up the catalytic particle was studied by transmission electron microscopy and circular dichroism. The reactive sites were designed with histidine residues to catalyze the hydrolysis of 2,4-dinitrophenyl acetate (DNPA). Enzymatic hydrolysis of DNPA was observed in the presence of the nanofibers by UV-vis spectroscopy and the Michaelis-Menten enzyme kinetics model. Significantly faster hydrolysis rate was observed in the presence of self-assembled nanofibers relative to spherical aggregates expected to have less order as well as single molecule catalysts. Copyright

Full text of DOI:10.1021/ja075044n

Published on Web 09/14/2007
A Self-Assembled Nanofiber Catalyst for Ester Hydrolysis
Mustafa O. Guler† and Samuel I. Stupp*,†,‡,§
Department of Chemistry and Institute for BioNanotechnology in Medicine, Department of Materials Science and
Engineering, and Feinberg School of Medicine, Northwestern UniVersity, 2220 Campus DriVe,
EVanston, Illinois 60208
Received July 8, 2007; E-mail: s-stupp@northwestern.edu
Chart 1. Chemical Structures of the Molecules Used to Create
The efficiency and selectivity of enzymes have always been a
source of inspiration to chemists working on the development of
robust synthetic catalysts. Mimicking enzymes with synthetic
materials has received considerable attention from both chemists
and biologists.1 Particles that could emulate enzymes have been
used to present reactive sites, for example, supramolecular systems
derived from polymers,2 nanostructures,3,4 and artificial proteins.5
Most of the synthetic particles investigated have spherical symmetry
and lack the internal order which is characteristic of proteins. The
use of imidazolyl groups in artificial catalysts has been popular
given the presence of histidine residues at the catalytic site of several
hydrolytic enzymes including acetylcholine esterase,6 chymot-
rypsin,7 trypsin,8 and kidney dialkylfluorophosphatase.9 A consider-
able increase of reaction rates has been observed upon aggregation
of active sites, presumably due to the increase in concentration of
reaction loci and cooperativity among functional groups.10
In the present study, we demonstrate hydrolysis of a model ester
on the surface of nanofibers formed by aggregation of peptide
amphiphiles. Peptide amphiphiles (PAs) have been previously
shown to self-assemble into nanofibers by hydrophobic collapse
of their alkyl tails and â-sheet formation among their peptidic
segments.11-14 We explored the ability of these cylindrical su-
pramolecular nanostructures for hydrolysis of 2,4-dinitrophenyl
acetate (DNPA). DNPA, a common model compound to study
catalytic activity of enzymes and synthetic systems,15 was selected
here as the substrate for esterase activity. Histidine residues were
incorporated into the peptide segment of PA molecules to mimic
hydrolytic enzymes discussed above. The compounds 1-4 (see
Chart 1) were prepared using solid-phase peptide synthesis (SPPS)
and purified by reverse phase HPLC. Compound 3 contains the
same peptide sequence KLLLAAA present in the PA structure with
histidine residues attached to the lysine residue as a peptide control
molecule without the palmitoyl group to eliminate the strong
amphiphilic character of the system responsible for the formation
of well-defined cylindrical aggregates. The proline residues were
incorporated into the peptide sequences of 2 and 4 in order to
prevent â-sheet formation and promote the formation of spherical
aggregates of PA molecules.17 Previous work has also demonstrated
that PA molecules containing proline residues readily assemble into
spherical aggregates.16 Compounds 2 and 4 were synthesized as
controls of nanostructure shape since cylindrical nanofibers are
characteristic structures in aggregated PA molecules that form
â-sheets.18
Nanostructures
their growth is electrostatically inhibited in acidic pH given the
positive charge on the molecules. The electrostatic barrier disap-
pears, and fibers grow spontaneously upon an increase of pH.
Transmission electron microscopy (TEM) micrographs of self-
assembled compound 1 at pH 7.4 revealed the formation of high-
aspect-ratio nanostructures with diameters of 7 ( 1 nm and lengths
ranging from hundreds of nanometers to several micrometers (see
Figure 1). At the same time, circular dichroism of PA 1 at pH 7.4
revealed a broad peak (n-π* transition) between 205 and 225 nm,
which is a signature for the predominant presence of â-sheets (see
Figure S1). On the other hand, compounds 2-4 were soluble at
pH 7.4 and did not form nanofibers but polydisperse spherical
aggregates instead with diameters ca. 15-20 nm observed by TEM
(see Figure 1). Also as expected, the CD spectra for compounds
2-4 revealed a peak (n-π* transition) between 200 and 205 nm
which can be attributed mainly to the presence of random coil
conformations in the peptide segment of these molecules (see Figure
S1).
Hydrolysis of DNPA by imidazolyl-functionalized molecules was
monitored by UV-vis spectroscopy at 400 nm to follow the
formation of 2,4-dinitrophenol at 25 °C and pH 7.4 in HEPES
buffer. The hydrolysis of phenyl acetates was previously reported
to be first-order in imidazole concentration.19 The imidazole directly
reacts with the phenyl acetate to form phenol and acylium ion-
imidazole complexes. The complex then reacts with water to form
acetic acid and imidazole. An imidazole moiety found in the
catalytic site of R-chymotrypsin is also known to activate ester
hydrolysis, and the Michaelis-Menten enzyme kinetics model was
used to calculate the reaction rate constant of the system.7,20 At
high substrate concentration, the catalyst-substrate complex is
present in excess in the media, so that the catalyst turnover becomes
the rate-limiting step in the enzymatic catalysis reactions. The
overall reaction rate is nearly insensitive to further increments in
substrate concentration.
Compound 1 was found to be soluble in water at pH 4 and
formed self-supporting gels at concentrations greater than 0.1 wt
% and pH above 6.5. Gel formation is a strong indication of self-
assembly of PA molecules into high-aspect-ratio nanofibers, and
The DNPA hydrolysis rates were calculated using varying
substrate conditions in the presence of a constant catalyst concentra-
tion. The Michaelis-Menten model was used to calculate the
hydrolysis rate constant for compound 1 because we observed
† Department of Chemistry and Institute for BioNanotechnology in Medicine.
‡ Department of Materials Science and Engineering.
§ Feinberg School of Medicine.
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J. AM. CHEM. SOC. 2007, 129, 12082-12083
10.1021/ja075044n CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
less internal order. A higher degree of order in the catalytic particle
should reduce the entropic penalty of binding events. Due to a high
density of reactive sites in spherical aggregates and possibly even
some short range order, hydrolysis of DNPA with 2 is slightly faster
than in the presence of 3, 4, His-Omet, and imidazole, which
suggests that increasing levels of internal organization in the catalyst
contribute to efficiency (see Table S2). In 3 and 4, the potent
entropy reducing effect of hydrophobic collapse by palmitoyl
segments is absent, and His-Omet and imidazole are of course
expected to function as non-supramolecular catalysts.
We conclude that the hydrolysis efficiency of DNPA, a model
ester compound, benefits by a high density of reactive sites
displayed on the surface of a supramolecular catalytic particle with
significant internal order. Considerably higher hydrolysis rate was
observed in the presence of internally ordered supramolecular
nanofibers as the catalytic particles, compared to catalysts in
solution and in spherical aggregates which should have less order.
The observations offer a new axis for the design of supramolecular
catalysts, suggesting that nanofibers of high aspect ratio and internal
order are potentially interesting catalytic devices. The function of
these nanostructures can be further increased taking advantage of
the possibility of co-assembling molecules in a nanofiber so that
various molecular recognition and chemical events can be integrated
into a single catalytic system.
Figure 1. Negatively stained TEM micrographs of 1 (A), 2 (B), 3 (C),
and 4 (D) at pH 7.4.
Acknowledgment. This work was supported by the U.S.
Department of Energy (Award No. DE-FG02-00ER54810) and the
National Institutes of Health (Award No. R01 EB003806-01). We
thank Steve Soukasene for TEM imaging, James Hulvat and Hakan
Usta for helpful discussions.
Supporting Information Available: Experimental details, mass
spectra, and hydrolysis rates. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 2. Observed rate increase in DNPA hydrolysis as the level of
molecular organization in the catalytic particles is varied.
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