Chemically Fueled Dissipative Self-Assembly that Exploits Cooperative Catalysis

Article abstract of DOI:10.1002/anie.201811749

In living systems, dissipative processes are driven by the endergonic hydrolysis of chemical fuels such as nucleoside triphosphates. Now, through a simple model system, a transient self-assembled state is realized by utilizing the catalytic effect of histidine on the formation and breaking of ester bonds. First, histidine facilitates the ester bond formation, which then rapidly co-assembles to form a self-supporting gel. An out-of-equilibrium state is realized owing to the cooperative catalysis by the proximal histidines in the assembled state, driving the second pathway and resulting in disassembly to sol. Cooperative effects that use the dual role of imidazoles as nucleophile and as proton donor is utilized to achieve transient assemblies. This simple system mimics the structural journey seen in microtubule formation where the substrate GTP facilitates the non-covalent assembly and triggers a cooperative catalytic process, leading to substrate hydrolysis and subsequent disassembly.

Full text of DOI:10.1002/anie.201811749

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Accepted Article
Title: Energy Dissipation of Chemical Fuel Driven Self-Assemblies via
Cooperative Effects
Authors: Subhajit Bal, Krishnendu Das, Sahnawaz Ahmed, and
Dibyendu Das
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201811749
Angew. Chem. 10.1002/ange.201811749
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10.1002/anie.201811749
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Chemically Fuelled Dissipative Self-Assembly exploiting
Cooperative Catalysis
Subhajit Bal, Krishnendu Das, Sahnawaz Ahmed and Dibyendu Das*
Abstract: In living systems, dissipative processes are driven by the
used. Notably, when we added a water soluble coupling agent,
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 4 mM) to
endergonic hydrolysis of chemical fuels such as nucleotide
triphosphates. Here we demonstrate through a simple model system, the mixture of NP and C18H, the solution rapidly became more
the realization of a transient self-assembled state by utilizing the
catalytic effect of histidine on the formation and breaking of ester
bonds. Firstly, histidine facilitates the ester bond formation, which
then rapidly co-assembles to form a self-supporting gel. Out of
equilibrium state is realized due to the cooperative catalysis by the
proximal histidines in the assembled state, driving the second
pathway and resulting in disassembly to sol. Cooperative effects
seen in natural and synthetic systems which use dual role of
imidazoles as nucleophile and as proton donor, is utilized here to
achieve transient assemblies. This simple system mimics the
structural journey seen in microtubule formation, where the substrate
viscous and turned into a self-supporting gel within 2 min. EDC
GTP facilitates the non-covalent assembly and triggers
a
cooperative catalytic process leading to hydrolysis of substrate and
subsequent disassembly.
Nature elegantly utilizes transient self-assembled structures that
are capable of exhibiting functions through energy consuming
processes.[1-3] These transient self-assembly processes rely on
the consumption of chemical fuels to perform reactions that are
thermodynamically unfavourable.[4-9] Chemical fuels are high
energy molecules which are kinetically stable and the stored
[10-19]
energy is utilized to drive biological processes.
One of the
classic examples of transient assemblies seen in Nature is in the
form of cytoskeleton proteins such as tubulins that self-assemble
to form microtubules.[18,19] Structurally, the individual tubulin
dimers assemble non-covalently using weak forces of
hydrophobic and electrostatic interactions in presence of
guanosine triphosphate (GTP) and the polymerization moves
towards equilibrium.[20, 21] Interestingly, in the assembled state
the catalytic potential of these individual tubulin dimers gets
significantly amplified resulting in hydrolysis of GTP to GDP
which causes conformation change and diassembly.[21,22]
Intrigued by this, we pursued to devise a simple system (Figure
1) which can undergo fuel driven self-assembly and utilize the
accelerated catalytic potential at assembled state to consume
the fuel. [21,22] To achieve this, we propose the use of cooperative
catalysis from proximal imidazole groups of histidine based
amphiphiles in assembled state (Figure 1). In this context, works
of Miravet/Escuder/Ulijn, Scrimin Lab and recent works of Prins
and co-workers have utilized cooperative catalysis at assembled
state in artificial systems.[22-27]
Figure 1. Schematic overview of fuel driven self-assembly and dissipation by
cooperative catalysis
has previously been used by Boekhoven and coworkers as the
high energy fuels for generation of transient supramolecular
materials.[29,30] Interestingly, after 30 min the gel showed
transition to a solution (Figure 2c) suggesting the formation of a
transient assembled state. Addition of EDC or NP separately did
not result in gelation. Varying the concentration of the coupling
agent and nucleophile resulted in different lifetimes of the
transient gels, reaching up to ca. 12 h (Figure 2b).
To investigate this transient behavior further, UV Vis
spectroscopy was used (Figure 3a). In case of C18H (12 mM)
with NP (4 mM), within two minutes of addition of EDC (4 mM),
we could see the generation of a low intensity peak at 268 nm
arising from the n to * transition of the generated ester C18H-
NP.[31] Notably, after 30 minutes, this peak gradually
disappeared indicating the transient existence of the high energy
The amphiphile (C18H) was synthesized by simply stearoylation
(C18) of histidine (Figure 2a).[28] C18H (12 mM) was dissolved in
aqueous solvent containing 20% v/v DMSO (See SI for details)
resulting in a free flowing solution. For the creation of the high
energy ester bond, 4-nitrophenol (NP) as the nucleophile was
ester C18H-NP. To investigate in
a control system, we
[*]
S. Bal, Dr. K. Das, Dr. S. Ahmed, Dr. D. Das
synthesized phenylalanine (F) analogue of compound C18H,
which was made following a similar process of reacting the
corresponding amino acid F with stearoyl chloride (C18F, Figure
Department of Chemical Sciences and Centre for Advanced
Functional Materials, Indian Institute of Science Education and
Research (IISER) Kolkata, Mohanpur 741246, India
E-mail: dasd@iiserkol.ac.in
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10.1002/anie.201811749
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2a). However, treatment with NP and EDC did not result in
generation of ester peak as presence of exogenous base is
required for the condensation of acid and alcohol. Addition of 4
mM N,N-diisopropylethylamine (DIPEA) resulted in the
generation of the peak at 268 nm suggesting formation of the
ester C18F-NP (Figure 3b). This peak plateaued after ca. 45 min
and no decrease of intensity of the peak could be seen even
after 10 h, suggesting higher stability of the kinetically stable
ester. Also, the fact that absence of exogenous base does not
result in any ester conversion, underpins the importance of the
built-in base in form of histidine’s imidazole of C18H. To
investigate further, we checked this dissipative system with
nuclear magnetic resonance (NMR) spectroscopy. Time
dependent 1H NMR of the sample containing the C18H in
morphology. Powder XRD was done with the dried powder and
xerogel of C18H solution and the transient gel respectively. The
xerogel samples of the transient gel showed crystalline
character with sharper peaks compared to only C18H (Figure
4d). Spacings of 4.38 Å and 3.9 Å suggest the distance between
two lipid layers of the amphiphiles and their sharper profiles
indicate closer packing distance.[32] Furthermore, rheological
measurements done with the transient gels showed superior
mechanical strength compared to the precursor acid C18H due
to the better packing in the transient coassembled gels (Figure
S2). The reason for the temporal existence of networks and gel
was likely due to cooperative catalysis in the assembled state.
This arises due to the proximity of the histidines in the closely
packed assembled nanostructures formed from the coassembly
of the precursor acid C18H with the transiently generated C18H-
NP ester. In extant biocatalysis, proximal localization of active
Figure 3. a) Time dependent absorption spectra of C18H with NP and EDC.
Inset shows the enlarged view of the absorption peaks at 268 nm. b) Change
of absorption at 268 nm with time. Time dependent 1H NMR spectra of c)
C18H and d) C2H with NP and EDC at 27 °C (R in the chemical structures
denote the rest of the molecular structure).
Figure 2. a) Chemical structures of molecules involved. b) Life time of
transient gels with varying concentration of EDC and NP or 4-methyl phenol c)
Vial images of C18H (12 mM) in presence of NP (4 mM) and EDC (4 mM) in
different time periods.
site residues remarkably facilitate the catalytic efficiencies. Also,
histidine dyads perform catalytic roles in hydrolases by shuttling
protons and influencing the local pH.[33] To investigate this
cooperative effect further, C2H was synthesized by simply
acetylating histidine amino acid (Figure 2a). Due to drastic
decrease of the tail hydrophobicity it was expected that C2H will
not access the self-assembled nanostructures and hence will not
show cooperative catalysis which is critical for dissipation in
these systems. Addition of NP and EDC to C2H also resulted in
the conversion to the corresponding ester which was followed at
268 nm (Figure 3b). The peak intensity did not show any
decrease over time, observed till 10 h. Also, from 1H NMR
spectroscopy, the peak intensity of marked protons (Ha, Hb,
yellow band in Figure 3d) of NP in the corresponding ester
remained unchanged with time with no indication of transient
nature. This mixture containing the C2H-NP ester and C2H acid
was also monitored by circular dichroism (CD) spectroscopy
which showed weak signals suggesting the absence of
assembled structures (Figure S3). In contrast, CD spectroscopy
done with transient ester of C18H analogue at ca. 10 min
showed stronger signals supporting the presence of
presence of NP and EDC indeed showed the generation of the
NP protons (yellow bands, protons marked Ha and Hb, Figure 3c)
of the ester. Notably, after 45 min, the signals of the NP
aromatic protons gradually decreased and eventually
disappeared completely showing the transient existence of the
ester (C18H-NP). In contrast, C18F showed presence of the
aromatic ester protons without any signs of decrease of peak
area with time till 10 h (Figure S1, Supporting Information). The
morphological transformations of the sample were checked at
different time periods by transmission electron microscopy (TEM,
Figure 4). Samples imaged immediately after mixing EDC, NP
with C18H, showed no network like morphologies with only
some aggregated structures (Figure 4a). Notably, after aging of
10 min, the formed transient gel showed dense networks with
extensive entanglements (Figure 4b). Interestingly, the same
sample which turned to solution after 30 minutes, showed
substantial decrease of fibrillar network structure (Figure 4c).
Control samples of C18H with or without addition of NP or EDC
separately, did not show any nanofibrillar network like
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supramolecular networks as observed from TEM (Figure 4).
TEM also revealed no network like morphologies for C2H-NP
containing sample (Figure S4). These results point that in the
coassembled state of C18H-NP and C18H, the proximal
histidines could be exhibiting cooperative effect. In this context,
if a less hydrolysable ester can be accessed, this out of
equilibrium state may be realized for longer time, a phenomenon
experimentally observed in case of microtubules.[20] Hence, 4-
methylphenol (MP) was used as the nucleophile to access the
kinetically more stable ester (Figure 2a, C18H-MP). Due to
replacement of electron withdrawing group of -NO2 with positive
inductive effect promoting group of methyl, the ester bond will be
less prone to hydrolysis in the assembled state. Indeed, addition
of 4-methylphenol along with EDC resulted in the partial
conversion to C18H-MP which formed a stable gel with life time
of ca. 3 days (Figure 2b). For support of cooperative catalysis,
rates of hydrolysis of ester were measured as a function of pH,
from 5.5 to 7.9 in buffer controlled conditions (Figure 4e).
Notably, the rate of the hydrolysis of the C18H-NP showed a bell
shaped dependence with highest activity at pH=6.5 which is in
the proximity of the pKa of the imidazoles (Figure 4e). This
suggests the dual cooperative role of the proximal imidazoles as
nucleophile and as proton-donor.34 At pH=7.9, base catalysed
rate of formation of the ester could be observed (Vi=1.11 µM
min-1).
C18H on its own does not form gel till 30 mM concentration. To
investigate whether the two components are involved in co-
assembly or self-sorting, CD spectra were recorded (Figure S6,
SI).35 Since C18H-NP is prone to hydrolysis, we chose the more
stable ester C18H-MP (Figure 1). The CD spectrum of C18H
showed a positive Cotton peak at 265 nm and was clearly
distinguishable from the spectrum of C18H-MP, which showed
two low intensity broad peaks at 290 nm and 322 nm (Figure
S6a, S6b). Notably, CD spectrum of 1:1 concentration mixture of
C18H and C18H-MP did not match with the spectrum obtained
from the simple sum (theoretical) of the CD traces of these
components (Figure S6c) suggesting co-assembled structures.
Further, the plot of CD intensity at =265 nm versus
concentration ratios of the components did not yield a linear
relationship suggesting the absence of self-sorted assembled
structures (Figure S7, SI).35, 36 To check whether the system can
be made reversible by addition of fuel again, EDC was mixed
after the gel to sol transition occurred. Addition of 4 mM of the
coupling agent resulted in the reformation of the self-supporting
gel which showed similar dissipative nature and showed
transition to sol in ca. 30 min. The cycle could be repeated for 3
times in total. Refueling on the fourth occasion did not result in
gelation presumably due to the accumulation of waste in form of
urea in the closed system. By monitoring the kinetics at
wavelength 268 nm, the generation and subsequent
disassembly was demonstrated through spectroscopy (Figure
4g).
In summary, we demonstrate a chemically fuelled transient self-
assembled system which dissipates energy from the assembled
state by utilizing cooperative catalysis. Lipid functionalized
histidine is used to facilitate both the reverse hydrolysis step in
disassembled form and augmented hydrolysis step due to
cooperative effects in the assembled state. This simple chemical
system mirrors the microtubule filament formation, where fuel
drives assembly and the accelerated catalytic activity at
assembled state results in degradation of fuel and
disassembly.21,27
Acknowledgements
Figure 4. a-c) Time resolved TEM images of C18H in presence of NP and
EDC. d) PXRD patterns of C18H and transient gel (xerogel). e) Rate of
hydrolysis of ester as a function pH 5.5 to 7 and the rate of formation of ester
DD is thankful to Nanomission Grant (SR/NM/NS-1082/2015),
DST, GOI for financial assistance. For fellowships, SB
acknowledges IISER Kolkata, KD acknowledges Nanomission
Grant, SA acknowledges NPDF (PDF/2017/000782), SERB.
at pH 7.9.
f) Time resolved conversion of C2H-NP and C18H-NP. g)
Reversible cycles of dissipative assembly-disassembly process fuelled by
repeated addition of EDC measured by monitoring absorbance at 268 nm.
Conflict of interest
To measure the extent of conversion of acid to ester, UV Vis
spectroscopy was used. From a standard plot created with
synthesized C2H-NP (Figure S5), the concentration of the
transient ester C18H-NP was calculated. Rapid conversion from
acid to ester with conversion of ca. 1.5 mM occurred in the first 2
minutes (Figure 4f). This corresponded to ca. 38 % of the NP
present (4 mM). The concentration of the ester gradually
decreased with time (Figure 4f). This result suggested that
coassembly of C18H and C18H-NP is responsible for the
creation of the transient gel. This finding from UV Vis
spectroscopy is supported by the observation from 1H NMR
spectroscopy (Figure 3c). Strikingly, the gel remains stable even
when only a fraction of transient ester (ca. 0.79 mM) is present
in the system after 30 min. It will be important to note here that
The authors declare no conflict of interest.
Keywords: Catalysis • Cooperative effects • Dissipative self-
assembly • Gels • Systems chemistry
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COMMUNICATION
Self-Driving: A simple fuel driven
Subhajit Bal,
self-assembled
system
is
Krishnendu Das,
Sahnawaz Ahmed and
Dibyendu Das*
demonstrated which achieves out
of equilibrium state by dissipating
energy from the assembled
structures. This is done utilizing
Page No. – Page No.
Chemically Fuelled
Dissipative Self-
Assembly exploiting
Cooperative Catalysis
cooperative
proximally
effects
located
of
the
histidines
which catalyse the hydrolysis of
ester bonds of the lipid tailed
amphiphile. This simple system
mirrors
observed in case of microtubule
filaments where augmented
the
dissassembly
catalysis at assembled state
results in energy dissipation and
subsequent disassembly.
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