PH clock instructed transient supramolecular peptide amphiphile and its vesicular assembly

Article abstract of DOI:10.1039/c9cc06934h

A new strategy to construct a transient supramolecular peptide amphiphile (SPA) and its vesicular aggregates is displayed. The construction of the amphiphile is assisted by the ternary complexation of cucurbit[8]uril and pH responsive imine bond formation. The transient assembly follows a pH clock set by urea/urease and hydrolysis of glucono delta-lactone (GdL). The transient assembly can be repeated for several cycles through feeding the system with the fuel (urea).

Full text of DOI:10.1039/c9cc06934h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
pH clock instructed transient supramolecular
peptide amphiphile and its vesicular assembly†
Cite this: Chem. Commun., 2019,
55, 14119
Payel Dowari,‡ Saurav Das, ‡ Bapan Pramanik
and Debapratim Das
*
Received 5th September 2019,
Accepted 29th October 2019
DOI: 10.1039/c9cc06934h
rsc.li/chemcomm
A new strategy to construct a transient supramolecular peptide short lived gels, vesicles, nano-reactors and nano-assemblies.2,3,10–17
amphiphile (SPA) and its vesicular aggregates is displayed. The An elegant approach toward developing transient systems is to
construction of the amphiphile is assisted by the ternary complexation temporally control factors such as temperature, pressure, light, pH
of cucurbit[8]uril and pH responsive imine bond formation. The etc.18,19 which mediate the formation of the self-assembled state.
transient assembly follows a pH clock set by urea/urease and Using a combination of two competing triggers that induce a
hydrolysis of glucono delta-lactone (GdL). The transient assembly temporary change in pH from acidic to basic reaching a transient
can be repeated for several cycles through feeding the system with state before reverting back to the acidic range can be established.20
the fuel (urea).
Combining this pH clock efficiently with any self-assembling system
whose assembly/disassembly is governed by the pH of the medium
Self-assembly of simple molecules into complex and highly can lead to a transient self-assembled system.3
ordered systems that perform vital biological processes has
been one of the principal tools used by nature for the creation to temporally form supramolecular peptide amphiphiles21 and
as well as sustenance of life.1 Most supramolecular systems are
under thermodynamic control and the self-assembly process is
generally an energetically down-hill process in which the stability
of the assembled state is conferred by the formation of a low-
energy equilibrium state.2–4 However, many self-assembly
processes occurring in nature such as the reversible formation
of actin filaments,5 formation of microtubules,6 cell division,6
motility7 and signal transduction8 are energetically demanding
processes and require a constant influx of energy in order to
retain the functional self-assembled state. The thermodynamic
pathway of assembly is inadequate to maintain the functional
efficacy of these systems and therefore such systems are kinetically
driven where the assembly process is governed by the assembly
pathway rather than the stability of the assembled state.9 Such out
of equilibrium self-assembly processes which are self-regulatory in
nature and generate supramolecular assemblies of short finite
lifetimes are referred to as transient self-assembly processes.
We anticipated that such a fuel-driven pH clock can be utilized
Recently, substantial efforts have been made to mimic the
out of equilibrium processes in nature and to unravel the
mysteries regarding the origin of life. The concept of transient
assembly has been exploited to develop temporally controlled and
Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039,
India. E-mail: ddas@iitg.ac.in
Scheme 1 Chemical structures of different components and graphical
presentation of the pH clock driven temporal formation of a CB[8] assisted
supramolecular peptide amphiphile and its vesicles.
† Electronic supplementary information (ESI) available: Experimental details,
characterization data and other supporting figures. See DOI: 10.1039/c9cc06934h
‡ These authors contributed equally.
This journal is ©The Royal Society of Chemistry 2019
Chem. Commun., 2019, 55, 14119--14122 | 14119

View Article Online
Communication
ChemComm
disappearance of the imine signals and re-appearance of the
aldehyde proton. The formation–deformation of the SPA can
thus be controlled using a pH switch.
To verify the proposed temporal aggregated system using
this SPA, the pH clock was established. The pH clock (Scheme 1)
has been realised by using a combination of two triggers: (i) the
autocatalytic reaction of urea with the enzyme, urease, that
releases NH3 and leads to an increase in pH of the system,
and (ii) the base-catalyzed saponification reaction of the dormant
deactivator Glucono delta-lactone (GdL) as a counter trigger
which furnishes gluconic acid and thereby decreases the pH.
Upon addition of a mixture of urea and GdL in a definite ratio to
an aqueous solution of the urease enzyme, initially a swift
increase in pH was observed. After the activation kinetics of
urease reaches its peak, no further rise in pH takes place. Under
alkaline conditions, the hydrolysis of GdL is aggravated and
initially, there is a rapid fall in pH until the pH reaches around
7.5 when the decrease in pH becomes slow and steady (Fig. 2A).
A probable reason for this is that as the optimum pH for urease
is 7.4,35 the rate of formation of ammonia abruptly increases in
this pH range and slows down the drop in pH. An increase in
the concentration of urea and/or urease not only elevates the
Fig. 1 (A) UV-Visible spectra of 0.75 mM aqueous solutions of different
components of 1 showing the appearance of the charge transfer band
upon ternary complexation in the case of 1. (B) Thermograms (top) and
binding isotherms (bottom) of MV-CHO@CB[8] with Nap-P at 298 K.
(C) 1H NMR of 1 and DA under different conditions showing the formation
and disappearance of the SPA as a function of pH of the medium.
also their self-assembly. In this regard, cucurbit[8]uril (CB[8], rate of activation but also causes an increment in the maximum
Scheme 1)22–24 assisted ternary complexation driven formation of overall pH. A high concentration of GdL restricts the rise in pH
supramolecular amphiphiles and their self-assembly into robust and escalates the rate of fall in pH. By fine tuning the rates of
and stimuli responsive vesicles have been reported on several the activation and deactivation steps, the lifetime of the tran-
occassions.25–28 Inspired by our previous experience with CB[8] sient supramolecular assemblies can be easily modulated. An
based vesicles, herein we report pH regulated transient for- aqueous solution containing 0.2 mg mLÀ1 of urease showed an
mation of a supramolecular peptide amphiphile (SPA) which initial pH in the range of 6.5–6.8. The pH of the solution was
self assembles to form transient vesicles.
adjusted to B5 before initiating the pH cycle. A very slow
Before constructing the pH clock and the transient system, deactivation kinetics was observed when the amount of GdL
the ternary complex (1) formation by a viologen unit (MV-CHO) was r2 equivalents of urea (Fig. 2A). This may be ascribed to
and a naphthalene functionalized peptide (Nap-P) with CB[8] the fact that one molecule of urea releases two molecules of
was confirmed by the appearance of the characteristic broad NH3, which neutralize the acid generated by GdL and thereby
charge transfer (CT) band at 396 nm in the absorption spectra
(Fig. 1A).29 The isothermal titration calorimetric (ITC) experiment
also showed a 1 : 1 complexation between Nap-P and MV-CHO@
CB[8] (Fig. 1B).30 Since a 1H NMR experiment with Nap-P resulted
in a complex spectrum, a shorter analogue, compound H,
(Scheme 1) was used to understand the situation. The NMR
spectra of different compositions of MV-CHO, H and CB[8] were
recorded (Fig. S1, ESI†). The up-field shifts31 of the MV- and
naphthalene-protons of H in the presence of CB[8] disclose a
slipped parallel stacking of the viologen and naphthalene units
inside the CB[8] cavity (detailed explanation is provided in the
ESI†). A similar slipped-stacking between donor acceptor groups
has already been reported.32,33 An ITC experiment using these
molecules also shows 1 : 1 : 1 ternary complexation (Fig. S2, ESI†).
In the presence of dodecylamine (DA), the ternary complex 1
formed the desired SPA under basic conditions. The formation
Fig. 2 (A) Optimizing the pH clock. The change in pH with time for four
different urea/GdL compositions. (B) Appearance of particles of different
sizes (from DLS) as a function of time for the transient system constructed
by 1 and DA in the presence of 0.2 mg mLÀ1 urease, and 1 : 3 urea/GdL
showing the response toward the pH clock. (C) FETEM, (D) FESEM and
(E) AFM images of the transient vesicles formed during the cycle
mentioned in (B).
of the SPA was confirmed by the disappearance of the aldehyde
signal and appearance of the imine peaks in the 1H NMR
spectra as shown in Fig. 1C. Imine bonds are dynamic covalent
bonds (DCBs) which are stable at basic pH but are highly labile
under acidic conditions.34 The SPA breaks down to the parent
amine and 1 upon acidification which is confirmed by the
14120 | Chem. Commun., 2019, 55, 14119--14122
This journal is ©The Royal Society of Chemistry 2019

View Article Online
ChemComm
Communication
slow down the decay in pH. In order to accelerate the deactivation
step, the amount of GdL used should be sufficient to counter the
neutralization caused by urea. Upon adding a mixture of urea and
GdL in the ratio 1 : 3, an initial pH jump to 9.2–9.3 followed by a
steady decay to 6.8–6.9 was observed in 120 minutes (Fig. 2A).
The kinetics of the transient formation of the SPA was then
assessed under the influence of the pH clock. A 0.1 mM
solution of the ternary complex, 1, in water containing
0.2 mg mLÀ1 of urease and an equivalent amount (0.1 mM)
of DA (dissolved in THF to facilitate solubilisation) showed an
initial pH of 7.0–7.2. The presence of the amine might be
responsible for this higher initial pH. The pH of the solution
was adjusted to B5 in order to avoid any SPA formation. The
Fig. 3 Three consecutive cycles of the transient SPA and its vesicle
formation as monitored by (A) emission spectra of DPH and (B) appearance
of particles of different sizes as a function of time (from DLS).
pH jump induced by the addition of urea and GdL (1 : 3) led to observed which confirms the vesicular nature of the system
the transient generation of the SPA by the formation of imine (Fig. S4, ESI†). Upon addition of HCl to this system, the
bonds under alkaline pH conditions.
emission returns back to its original position. Furthermore, to
The aggregation process of the so formed SPA was monitored confirm the transient nature, similar experiments were per-
using Dynamic Light Scattering (DLS) measurements. Fig. 2B formed (1 + DA + DPH + urease) in the presence of GdL-urea
shows the appearance of particles of different sizes and change and a prominent enhancement in the emission of DPH was
in pH of the system as a function of time. Prior to the addition observed until 5 min followed by a decay to its original position
of the triggers, no particular size distribution was observed in within 90 min (Fig. 3A).
the DLS profile. However, aggregates with a hydrodynamic
It is worth mentioning that, control experiments in the
diameter of B220 nm started (Fig. S3, ESI†) appearing soon absence of CB[8] showed the formation of imine bonds at higher
after the pH clock started (addition of urea/GdL). Peaks corres- pH. However, neither any size distribution nor any particular
ponding to 220 nm distribution continued appearing until the morphology could be found for this system. The attachment of
pH of the system remained above 7.4 (B60 min). However, after DA with MV-CHO certainly created an amphiphile which pre-
that, the distribution disappeared completely. Even though the sumably could not form any vesicular aggregate as no particular
pH jump takes place almost instantaneously, aggregation takes morphology could be seen from microscopic experiments. Experi-
place in a relatively slower and delayed fashion. This might be due ments with DPH also showed no change in the emission of the
to the reason that the ammonium ions present in the system dye upon increasing the pH from 5 to 8 confirming the absence
might compete with DA for imine formation.3 However, DA, owing of vesicles (data not shown). It is well-established that asym-
to its stronger hydrophobic interactions in the assembled state, metric viologen amphiphiles are not capable of forming a
forms stronger imine bonds and outweighs the effect of ammonium vesicular assembly alone but ternary complexation with CB[8]
ions. The aggregation pattern clearly demonstrates a pH dependent assists the aggregation to vesicles.36
behaviour.
Confocal laser scanning microscopy (CLSM) was performed
In order to determine the morphology of the supramolecular to directly visualize the assembly as well as the disassembly
aggregates, microscopic techniques were employed. The FETEM processes. A hydrophobic dye, Nile red (NR) was used to facilitate
image of the sample at pH 7.8 shows hollow spherical aggregates the visualization of the aggregates. A control experiment with 1
with a thin dark boundary which is indicative of the formation of and DA at pH 8 showed vesicle like structures under the micro-
vesicles (Fig. 2C). The diameter of the vesicles was found to be scope (Fig. S5, ESI†). The fluorescent spots remained unchanged
200 nm which is in good agreement with the DLS data obtained. for 2 h ensuring no photo-bleaching. During the experiment
Furthermore, the absence of any specific morphology for the under the pH clock, initially, no fluorescence was observed in the
samples at pH below 7.4 corroborates the transient formation of absence of pH triggers due to the absence of any vesicles (Fig. 4).
vesicles. FESEM and AFM experiments also showed the formation However, the fluorescence intensity of NR increased abruptly
of spherical aggregates with similar dimensions (Fig. 2D and E).
after 15 minutes due to the addition of the fuel indicating the
Ternary complexation induced formation of supramolecular formation of vesicles and entrapment of NR. As the reaction
amphiphiles and their vesicles are already demonstrated by our proceeds further, the fluorescence intensity diminishes slowly
group and in the present case, the microscopic images also indicating the breakdown of the vesicles. One cycle of vesicle
indicate the formation of vesicles.27 However, it is important to formation and dissociation was visualized by recording an image
confirm that the spherical aggregates are indeed vesicles. To every 30 s. Time lapsed movies for one such cycle have been
prove the vesicular nature of the aggregates, diphenylhexatriene provided as Videos S1 and S2 (ESI†). The number of fluorescent
(DPH) was used which is well-known for the enhancement of objects (Fig. S6, ESI†) was then counted to quantify the analyses.
emission upon encapsulation in the lipid bi-layer of vesicles.2 In From the movies, Fig. 4 and Fig. S6 (ESI†), the formation and
the present case, when the pH of the system (1 + DA) is dissociation of the vesicles is clearly visible as the number of
increased to B8 by the addition of NaOH solution, significant fluorescent particles increased and then disappeared following
enhancement in the emission (at 438 nm; lex = 355 nm) was the pH clock.
This journal is ©The Royal Society of Chemistry 2019
Chem. Commun., 2019, 55, 14119--14122 | 14121

View Article Online
Communication
ChemComm
DD acknowledges financial support from SERB (EMR/2016/
000857), India, and the UKIERI (DST/INT/UK/P-119/2016), India.
Conflicts of interest
There are no conflicts to declare.
Notes and references
1 D. J. Kushner, Bacteriol. Rev., 1969, 33, 302–345.
2 S. Maiti, I. Fortunati, C. Ferrante, P. Scrimin and L. J. Prins, Nat.
Chem., 2016, 8, 725.
3 A. Jain, S. Dhiman, A. Dhayani, P. K. Vemula and S. J. George, Nat.
Commun., 2019, 10, 450.
4 J. Wang, K. Liu, R. Xing and X. Yan, Chem. Soc. Rev., 2016, 45, 5589–5604.
5 E. Reisler and E. H. Egelman, J. Biol. Chem., 2007, 282, 36133–36137.
6 A. Desai and T. J. Mitchison, Annu. Rev. Cell Dev. Biol., 1997, 13, 83–117.
7 A. J. Howard and R. R. L. Clark, Appl. Mech. Rev., 2002, 55, B39–B39.
8 S. O. Rizzoli, EMBO J., 2014, 33, 788–822.
Fig. 4 CLSM images of the transient system constructed by 1, DA,
0.2 mg mLÀ1 urease, and 1 : 3 urea/GdL in the presence of NR at different
times showing the temporal formation of the vesicles.
9 E. Mattia and S. Otto, Nat. Nanotechnol., 2015, 10, 111.
10 S. De and R. Klajn, Adv. Mater., 2018, 30, 1706750.
11 M. Kumar, N. L. Ing, V. Narang, N. K. Wijerathne, A. I. Hochbaum
and R. V. Ulijn, Nat. Chem., 2018, 10, 696–703.
12 S. Bal, K. Das, S. Ahmed and D. Das, Angew. Chem., Int. Ed., 2019, 58,
244–247.
13 H. Wang, Y. Wang, B. Shen, X. Liu and M. Lee, J. Am. Chem. Soc.,
2019, 141, 4182–4185.
14 F. della Sala, W. Verbeet, S. Silvestrini, I. Fortunati, C. Ferrante and
L. J. Prins, ChemNanoMat, 2018, 4, 821–830.
15 J. Boekhoven, W. E. Hendriksen, G. J. M. Koper, R. Eelkema and
J. H. van Esch, Science, 2015, 349, 1075–1079.
16 J. P. Wojciechowski, A. D. Martin and P. Thordarson, J. Am. Chem.
Soc., 2018, 140, 2869–2874.
17 G. Wang, B. Tang, Y. Liu, Q. Gao, Z. Wang and X. Zhang, Chem. Sci.,
2016, 7, 1151–1155.
18 S. Panja, C. Patterson and D. J. Adams, Macromol. Rapid Commun.,
2019, 40, 1900251.
Having established the transient formation of vesicles, we
were interested to check the repeatability of the transient
assembly by refuelling after each cycle. Refuelling after the first
cycle reproduced similar cycles of events and the aggregation in
subsequent cycles was studied using DLS and fluorescence
spectroscopy (using DPH) as shown in Fig. 3A and B. In
subsequent cycles, a lower rise in initial pH followed by a rapid
decay in pH was observed (Fig. S7, ESI†). The DLS data also
showed a steady decrease in the lifetime of the assembled state
in consecutive cycles. This fall in efficiency of the pH clock is
due to the accumulation of gluconic acid in the system after
each cycle which increases the overall acidity of the system.
Fluorescence studies using DPH as the hydrophobic probe also
showed an increase in the fluorescence intensity followed by a
steady decay. The second reaction cycle shows a nearly identical
variation in the fluorescence intensity which indicates the
stability of the system under repeated pH cycles.
¨
19 P. K. Kundu, D. Samanta, R. Leizrowice, B. Margulis, H. Zhao, M. Borner,
T. Udayabhaskararao, D. Manna and R. Klajn, Nat. Chem., 2015, 7, 646.
20 T. Heuser, A.-K. Steppert, C. M. Lopez, B. Zhu and A. Walther, Nano
Lett., 2015, 15, 2213–2219.
21 A. Dasgupta and D. Das, Langmuir, 2019, 35, 10704–10724.
To gain further insight into the dynamics of the aggregation, 22 K. I. Assaf and W. M. Nau, Chem. Soc. Rev., 2015, 44, 394–418.
23 S. K. Steven, J. Barrow, M. J. Rowland, J. del Barrio and O. A. Scherman,
DLS studies were performed with aliphatic amines of varying
Chem. Rev., 2015, 115, 12320–12406.
chain lengths (Fig. S8, ESI†). The chain length had no significant
24 D. Das, K. Assaf and W. Nau, Front. Chem., 2019, 7, 619.
effect on the aggregation behaviour and time scale. However, a 25 Y. J. Jeon, P. K. Bharadwaj, S. Choi, J. W. Lee and K. Kim, Angew.
Chem., Int. Ed., 2002, 41, 4474–4476.
26 D. Jiao, J. Geng, X. J. Loh, D. Das, T.-C. Lee and O. A. Scherman,
direct proportionality between the size of the aggregates and the
chain lengths could be observed as the average diameter increases
Angew. Chem., Int. Ed., 2012, 51, 9633–9637.
with the chain length of the amine.
In conclusion, we have shown the transient formation of a
SPA which subsequently forms a vesicular assembly in response
27 J. H. Mondal, S. Ahmed, T. Ghosh and D. Das, Soft Matter, 2015, 11,
4912–4920.
28 J. H. Mondal, T. Ghosh, S. Ahmed and D. Das, Langmuir, 2014, 30,
11528–11534.
to a pH clock. The formation of the SPA is assisted by the 29 Y. H. Ko, E. Kim, I. Hwang and K. Kim, Chem. Commun., 2007,
1305–1315.
ternary complexation by CB[8]. The pH clock is established by
combining urea/urease based fast biocatalytic processes and
30 M. E. Bush, N. D. Bouley and A. R. Urbach, J. Am. Chem. Soc., 2005,
127, 14511–14517.
slow hydrolysis of GdL. The imine bond formation under basic 31 Y.-M. Zhang, J.-H. Liu, Q. Yu, X. Wen and Y. Liu, Angew. Chem., Int.
Ed., 2019, 58, 10553–10557.
32 B. Pramanik, S. Ahmed, N. Singha, B. K. Das, P. Dowari and D. Das,
conditions and its hydrolysis under an acidic medium are
the key chemical transformations used for the construction of
Langmuir, 2019, 35, 478–488.
the SPA and its transient assembly. The temporal formation of the 33 N. Singha, B. K. Das, B. Pramanik, S. Das and D. Das, Chem. Sci.,
2019, DOI: 10.1039/C9SC03417J.
34 B. Pramanik and D. Das, J. Phys. Chem. C, 2018, 122, 3655–3661.
35 S. D. Cesareo and S. R. Langton, FEMS Microbiol. Lett., 1992, 99, 15–21.
vesicle requires chemical fuel to maintain the out-of-equilibrium
state and several cycles of the transient assembly can be achieved by
supplying the fuel to the system.
36 J. H. Mondal, S. Ahmed and D. Das, Langmuir, 2014, 30, 8290–8299.
14122 | Chem. Commun., 2019, 55, 14119--14122
This journal is ©The Royal Society of Chemistry 2019