Protein and enzyme gated supramolecular disassembly

Article abstract of DOI:10.1021/ja4108676

An amphiphilic nanoassembly was designed to respond to the concurrent presence of a protein and an enzyme. We present herein a system, where in the presence of these two stimuli supramolecular disassembly and molecular release occur. This molecular releas

Full text of DOI:10.1021/ja4108676

Communication
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Open Access on 01/21/2015
Protein AND Enzyme Gated Supramolecular Disassembly
Jing Guo, Jiaming Zhuang, Feng Wang, Krishna R. Raghupathi, and S. Thayumanavan*
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States
S
* Supporting Information
ABSTRACT: An amphiphilic nanoassembly was designed
to respond to the concurrent presence of a protein and an
enzyme. We present herein a system, where in the
presence of these two stimuli supramolecular disassembly
and molecular release occur. This molecular release arises
in the form a fluorescence response that has been shown
to be specific. We also show that this system can be
modified to respond only if light stimulus is also present in
addition to the protein and the enzyme. Demonstration of
such supramolecular disassembly principles could have
broad implications in a variety of biological applications.
upramolecular assemblies that disassemble and release
molecules due to a change in their environment have been
S
Figure 1. Schematic representation of protein AND enzyme gated
supramolecular disassembly.
of interest in a variety of areas including sensing, diagnostics,
catalysis, and drug delivery.1−4 The supramolecular disassembly
is often the result of a chemical change that occurs in the key
functional groups of the assembly in response to the change in
the environment, often referred to as the stimulus. Selectivity in
this stimulus responsive disassembly is important for most of
the targeted applications. A versatile approach to enhancing
selectivity involves the imposition that the disassembly will
occur only in the presence of two or more stimuli. This is
reminiscent of molecular AND gates, where systems involving
fluorescence sensing have been previously demonstrated.5
These systems were mainly focused on changing a non-
fluorescent small molecule to a fluorescent one or vice versa in
response to the concurrent presence of two stimuli. These
systems are useful in certain sensing and chemical logic gate
applications, but are not easily extended to protein-based
systems as they are mostly demonstrated in apolar solvents.
Supramolecular disassembly due to the presence of two or
more stimuli, especially involving polymeric assemblies in
aqueous phase, is also known.6 However, these systems have
mainly focused on stimuli such as light, pH, redox conditions,
and temperature.7−10 While these stimuli have been of interest
due to their implications in biology, the imbalances in these
factors in biology are considered to be secondary indicators.
The primary indicators of biological imbalances, and thus
diseased tissue locations, are variations in protein concen-
trations or enzymatic activity. Therefore, protein-responsive
disassembly has been of recent interest.11 Considering the
decisive advantages of multistimuli responsive assemblies, we
were interested in addressing the challenge of designing a
system that not only responds to proteins but also would
respond only due to the concurrent presence of two different
proteins. We demonstrate a versatile new molecular design that
allows for such a supramolecular disassembly, illustrated in
Figure 1. We also further demonstrate that the same
supramolecular system can be modified to include a third
AND input to generate the molecular release response.
The molecular design strategy that has the potential to serve
as a protein-based AND logic gate is shown in Figure 1. We use
a facially amphiphilic molecule to test this design concept. In an
amphiphilic micelle-like assembly, the hydrophobic units are
buried in the core of the assembly and therefore should not be
accessible to an enzyme. For this to be true, it is important that
the equilibrium between the monomeric state of the
amphiphilic molecule and its aggregated state heavily favors
the latter (represented by equilibrium a in Figure 1). We have
previously shown that micelle-like supramolecular aggregates,
from facially amphiphilic molecules, can be disassembled due to
a ligand−protein binding event.11c The disassembly occurs due
to the change in the hydrophilic−lipophilic balance caused by
the protein binding; i.e., the protein binding causes the
equilibrium to shift to the monomeric state of the amphiphile
(represented as equilibrium b in Figure 1). We hypothesize
then that by incorporating an enzyme-sensitive linker in the
hydrophobic part of the amphiphile, we will favor the
aggregated state in the absence of the protein, but favor the
monomeric state in the presence of the protein. This should
generate a system that would release a covalently bound
molecule only in the presence of both protein stimuli
(determined by the difference in steps (a,c) vs (b,d) in Figure
1).
Received: October 28, 2013
Published: January 21, 2014
© 2014 American Chemical Society
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a,b). Probing these by transmission electron microscopy
(TEM) showed that these molecules indeed form spherical
assemblies (Figure S2c). We attempted to assess the critical
aggregation concentration (CAC) of 1 using guest encapsula-
tion measurements without success. We presumed that this
could be due to its rather low CAC. Therefore, we resorted to
surface tension measurements, and there was no obvious
change in the surface tension, compared to pure water within
concentration range measured (Figure S2d). These results were
considered to suggest that the equilibrium strongly favors the
aggregated state in 1 and that its CAC is very low.
Scheme 1. Synthesis of Target Amphiphile Molecule 1
The stimuli responsive characteristics of this assembly were
then studied in the presence of the protein, enzyme, and the
combination using the release of the fluorescent coumarin as
the probe. Figure 2a shows the results of this study using a 13
To test this design hypothesis, we targeted the amphiphilic
molecule 1 shown in Scheme 1. In this molecule, the
dinitrophenyl ligand (complementary and specific to anti-
DNP immunoglobulin G (IgG)) molecule is presented at the
hydrophilic face of the biaryl unit in 1. Similarly, our design
requires that the enzyme-sensitive unit be placed at the
hydrophobic part of 1. We chose the acetal-functionalized
coumarin ester as the hydrophobic moiety, because (a) this
functional group is capable of being cleaved by an esterase and
(b) the product of the enzymatic reaction with this substrate is
a highly fluorescent coumarin, which allows for directly
monitoring the enzyme-induced molecular release. Note that
the coumarin moiety is presented at all three repeat units in 1,
as this feature provides the key to introducing a third stimulus
into this process (vide infra). Moreover, the presence of the
pro-fluorophore in all three units also will produce the optimal
fluorescence in response to the presence of the protein and the
enzyme.
The target molecule 1 was synthesized through a multistep
modular synthesis. Briefly, the alkyne-based precursor molecule
4 was achieved by reacting the amphiphilic benzyl bromide
molecule 2 to amphiphilic biaryl unit 3 containing a 2,4-
dinitrophenol (DNP) ligand using an alkylation reaction, as
shown in Scheme 1. The synthetic steps involved in obtaining
the amphiphilic repeat units themselves are outlined in the
Supporting Information. The alkyne precursor molecule 4 was
reacted with the coumarin azide 5 under the Huisgen 1,3-
dipolar cylcoaddition reaction conditions, the so-called ‘click
chemistry’, to yield the target amphiphile 1.
Figure 2. Disassembly and coumarin release from 1 (13 μM) in the
presence of anti-DNP IgG (1 μM) and PLE (50 nM): (a)
Fluorescence change with time. (b) Size change after 8 h, monitored
by DLS.
μM solution of 1 in the presence and absence of anti-DNP IgG
and porcine liver esterase (PLE). We chose this concentration
for 1, since similar amphiphilic molecules exhibited CACs
slightly below this.11a,c,e First, no fluorescence was generated in
the absence of the stimulus (anti-DNP IgG or PLE) or in the
presence of anti-DNP IgG. With PLE, there was a small amount
of fluorescence generated, indicating that equilibrium a (Figure
1) significantly favors the aggregated state of the amphiphile.
However, in the presence of both anti-DNP IgG and PLE, there
was rather rapid fluorescence generation in the solution
indicating that anti-DNP IgG influences the equilibrium to
favor the monomeric state in equilibrium b. The initial rate of
ester cleavage in the presence of anti-DNP IgG and PLE was
found to be about 26 times faster than that in the presence of
PLE alone. The stimuli-induced disassembly process was also
monitored by DLS. Figure 2b shows that the size of the
assembly does not change in the absence of either of the
proteins or in the presence of PLE after 8 h. However, the size
drastically changed in the presence of anti-DNP IgG, again
confirming the dominance of the monomeric state of the
amphiphile in solution (equilibrium b in Figure 1). Under-
standably, the size of the assembly was also small in the
presence of both anti-DNP IgG and PLE.
Following the synthesis of the molecule 1, we characterized
the amphiphilic assembly formed by this molecule in aqueous
phase. Measuring the size of the assembly using dynamic light
scattering (DLS) revealed that the size of the aggregates formed
from these molecules was about 480 nm, even at concentrations
as low as 1 μM. We have characterized these assemblies as
micelle-like aggregates, because these are not simple micelles.
However, analogous assemblies do have a hydrophobic core
and are solid structures.11a,c,e,12 The assembly size was
monitored for a 24 h time period, and the size of the assembly
in solution did not change over this time period (Figure S2
In order to examine if the cooperative disassembly and
molecular release is specific to the anti-DNP IgG/PLE
combination, we studied coumarin release in the presence of
PLE and other proteins, viz. pepsin (anionic, pI 2.2−3.0),
chymotrypsin (cationic, pI 8.8), and thrombin (neutral, pI 7.1).
Figure 3a shows that only the anti-DNP IgG/PLE combination
generated the fluorescence response. The responses in the
presence of other proteins were indistinguishable from the PLE
itself (Figure 2a). Moreover, DLS shows that disassembly does
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Communication
combination causes the assembly to increase in size, likely due
to the aggregation caused by the anti-DNP IgG binding (Figure
4b). After de-crosslinking however, disassembly indeed occurs
in the presence of anti-DNP IgG.
In summary, we have shown the following: (i) by designing
an appropriate amphiphilic molecule containing a protein-
specific hydrophilic ligand moiety and enzyme-specific hydro-
phobic substrate moieties, supramolecular assemblies that
exhibit molecular release in the presence of both the
complementary protein and the enzyme, but not in the
presence of either of these stimuli alone, can be achieved; (ii)
the dual protein-responsive molecular release is due to the
variation in the monomer−aggregate equilibrium in the
presence and absence of the complementary protein; (iii) a
fluorescent signal can be generated by incorporating a pro-
fluorophore as the hydrophobic enzyme substrate; (iv) the
fluorescence response is specific to the protein/enzyme
combination; (iv) by locking the aggregated state of the
equilibrium, the protein response characteristics can be turned
off, which can then be reversed by de-crosslinking the
aggregate, thus making the system responsive to a third
stimulus input. The supramolecular principles, developed here,
are likely extendable to a variety of other stimuli and thus have
potential applications in a variety of areas including delivery and
sensing, which are the foci of current work in our laboratories.
Figure 3. Studies of binding specificity enhanced dye release. (a)
Fluorescent intensity change upon time. (b) Size after 8 h, monitored
by DLS.
not occur in the presence of any of these proteins once again
supporting the specificity of the disassembly and molecular
release to the anti-DNP IgG/PLE combination (Figure 3b and
Figures S5, S6 also). To further test this specificity, we
investigated a structurally similar molecule that lacks the ester
substrate and the DNP ligand moiety, which showed no change
in size with the presence of anti-DNP IgG (Figure S4a, b). We
also tested dye release caused by nonspecific binding protein:
IgG from rat serum. Nonspecific binding protein IgG
incorporation with PLE afforded a small increase in guest
release in comparison to PLE alone, which indicates that
specific binding is required for the dramatic guest release
observed with anti-DNP IgG (Figure S4c, d).
Finally, we were interested in exploring whether we can
introduce a third, non-protein input into this scheme. We
hypothesized that crosslinking the assemblies will lock the
aggregated state of the molecule and thus not allow for binding
induced disassembly (step f, Figure 1). Then, de-crosslinking
this aggregate in step e using a third stimulus will be an essential
step to access the binding induced monomeric state of
equilibrium b, as shown in Figure 1. To examine this possibility,
the hydrophobic coumarin units in the assembly were photo-
crosslinked at 365 nm irradiation.13 Figure 4a shows that
ASSOCIATED CONTENT
* Supporting Information
Synthetic and characterization details. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
thai@chem.umass.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank NIGMS of the National Institutes of Health (GM-
065255) for support. We thank the NIGMS of the National
Institutes of Health for a CBI fellowship to J.Z. (T32
GM08515).
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