Adenosine-Phosphate-Fueled, Temporally Programmed

Aug 28, 2017 - Direct visualization and real-time monitoring of dissipative self-assembly by synchronously coupled aggregation-induced emission. Wen-C...
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Cite This: J. Am. Chem. Soc. 2017, 139, 16568-16575

Adenosine-Phosphate-Fueled, Temporally Programmed Supramolecular Polymers with Multiple Transient States Shikha Dhiman, Ankit Jain, Mohit Kumar, and Subi J. George* Supramolecular Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore, India 560064 S Supporting Information *

ABSTRACT: Natural systems have been an inspiration to synthetic supramolecular chemistry. Synthetic demonstrations of dissipative biological systems such as actin filaments are a formidable scientific challenge in attaining future life-like materials. Dynamic instability of such structures beckons control of self-organization in the temporal regimes. In this study, we present a fueldependent helical assembly of a supramolecular polymer. We further attempt the synthetic manifestation of a temporally programmable self-assembly. Additionally, the fuel-induced chiral (re)organization with the employment of various enzymes singularly and in tandem have resulted in designing a multistate transient self-assembly. These parameter modulations result in controllable lifetimes and rates. We thus report, for the first time, a temporally programmed multistate reorganization of self-assembly.

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INTRODUCTION Naturally occurring systems are omnipresent inspirations for supramolecular chemistry.1 Although passive systems based on supramolecular interactions that mimic biological structures have been extensively investigated, active systems with temporal control for life-like materials persists as an intriguing challenge.2 There have been attempts to obtain kinetically trapped assemblies that convert, with time, to more stable thermodynamic assemblies that are highly dependent on the pathway of preparation.3 Although the conversion of these kinetically trapped/metastable assemblies to thermodynamic assemblies occurs over a certain time period, a temporal programming of this transformation is not efficiently possible as they are trapped by an energy barrier. Thus, achieving a temporal control over self-organization is the key to bridge the link between current dynamic supramolecular systems and future nonequilibrium materials with adaptive and self-repairing characteristics that can then be time-coded for various functions.4 The temporal control obtained by natural systems is a consequence of their fuel-driven functioning under nonequilibrium.5,6 Naturally occurring self-assembled cytoskeleton proteins such as microtubules and actin filaments perform invaluable functions of cell division and cell mobility by undergoing a transient change in their degree of polymerization, i.e., length of the filament/ tubule.6 Further, these natural systems are fueled by biomolecules that bear the energy expense to work at out-ofequilibrium conditions by undergoing chemical modifications. Most of the biological systems including actin filaments and transmembrane proteins such as ATP-binding cassette (ABC) transporters7 utilize adenosine triphosphate (ATP) as the biofuel to drive the functions, and thus, it is known as energy currency of the cell.8 © 2017 American Chemical Society

Taking inspiration from natural systems to create materials with spatiotemporal control over their organization as well as functions, new approaches and strategies in supramolecular chemistry have been attempted.9a The two important fuels, light and chemical reagents, have been utilized in various approaches to build up transient nanostructures.9 However, chemically fueled systems are the basis of most of the naturally occurring transient systems. Hence, a chemically fueled bioinspired approach is thought to be the key to achieve synthetic nonequilibrium systems.10 One of the earliest example of transient self-assembly has been presented by van Esch’s group, where a methylating agent fuels transient selfassembly of carboxylate-appended monomers.11 An alternative enzymatic approach by Ulijn and coworkers shows the use of αchymotrypsin enzyme to form and disintegrate the smallpeptide-assembling molecule, thereby presenting a biocatalytic12 transient gelation.13 Walther’s group reported a unique nonlinear temporal control over pH change using an urease enzyme. This urease enzyme can be coupled to any pHsensitive system to transiently modulate its activity. They further employed this temporal control over pH change to transiently program gelation of molecules, conformation of imotif DNA, and control the photonics behavior of polymers.14 In a very interesting bioinspired approach, Prins and coworkers employed ATP, the biofuel, to drive the formation of vesicular nanoreactors in the presence of an ATP-hydrolyzing enzyme, showing an unprecedented example of temporal control over reaction kinetics via transient formation of nanoreactors.15 They have also modified a zinc-receptorReceived: July 18, 2017 Published: August 28, 2017 16568

DOI: 10.1021/jacs.7b07469 J. Am. Chem. Soc. 2017, 139, 16568−16575

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Scheme 1. Schematic Representations of Investigated Molecular Structures, Various Modes of Self-Assembly, and Selected Routes of Enzymatic Action†

† (a) Molecular structures of NDPA adenosine phosphates (AXP), phosphocreatine (PCr), and creatine (Cr). (b) Schematic representation of transient self-assembly of NDPA with fuel-dependent and enzyme-mediated interconversion of assemblies depicting a transient self-assembly with multistate helical conformational changes. (c) Graphical representation of transient self-assembly with multi-nonequilibrium conformational state depicted in growth and conformation vs time. In (b) and (c), the peach background represents right-handed (P) helical assembly, blue represents left-handed (M) helical assembly, and gray represents disassembled/racemic state. Arrows in (b) and (c) indicate the addition or transformation of various fuels. The figure has NDPA as a monomer in initial state; however, we are uncertain about its extent of assembly and racemic or achiral structure and, thus, keep it as a NDPA monomer. (d−f) Schematic representation of enzymatic action on their substrates with rate constants of steps: (d) Alkaline phosphatase (ALP), (e) Apyrase (AP), (f) Creatine phosphokinase (CPK).

functionalized gold-nanoparticle system16 via a similar strategy to show transient signaling.17 Very recently, our group has investigated the conformational characteristics of temporally controlled supramolecular polymers18 and introduced the concept of “transient helicity” fueled by ATP via an “enzyme in tandem” approach to uniquely control rate of formation and decay of transient helical conformations.18a,19 In a very recent report, Hermans and co-workers extended transient selfassembly using a dialysis membrane setup to control influx of fuel and outflux of waste to obtain nonequilibrium steady states.20 Although synthetic systems reported so far represent exceptional examples in supramolecular transiency, advancement toward higher complexity of transient systems with multiple connected networks, which are ubiquitous to nature, needs to be addressed. Moreover, chirality is an important characteristic of natural systems and actin filaments that undergo a chiral helical transient state. However, reports highlighting chirality of the transient assembly are scarce.18a,20 Herein, we attempt to obtain the best of both worlds by obtaining a transient self-assembly of a helical supramolecular polymer, which can adopt multiple nonequilibrium conformational (helical) states en route to disassembly, via an interconnected network of nonequilibrium states. These are mediated by enzymes and programmed by chemical fuels (adenosine phosphates). Our group has been working on adenosine-phosphate-driven chiral supramolecular polymerization of chromophores appended with phosphate-receptor group zinc(II) dipicolylethylenediamine (Zn−DPA) in an attempt to mimic fuel-triggered self-assembly of biological molecules.18a,19,21,22 We have previously shown that the naphthalene diimide derivative

with Zn−DPA receptors, NDPA, self-assembles into onedimensional fibers on interaction with adenosine phosphate fuels. We have shown that these assemblies undergo adenosinephosphate-selective chiral organization (Scheme 1a,b).19 In this study, we further investigate the degree of polymerization of these adenosine-phosphate-induced assemblies to construct a transient supramolecular polymer. The chemical fuels utilized in this study are phosphate guests, which can be further derivatized according to our requirements. Moreover, these fuels are biological molecules, which makes the system highly modular, and hence, we can utilize naturally occurring enzymes singularly and in tandem to bring out a variety of fuel transformations via different pathways, as a consequence of which the self-assembly of NDPA will undergo transformations (Scheme 1a−c) thus making our system unique and modular. The enzymes used here are alkaline phosphatase (ALP), apyrase (AP), and creatine phosphokinase (CPK) (Scheme 1d−f, Figures S1,S2). Alkaline phosphatase hydrolyses the phosphoanhydride bond unselectively, whereas apyrase has a 10:1 ATPase to ADPase ratio that results in a higher rate of ATP hydrolysis than that of ADP hydrolysis. In contrast, creatine phosphokinase catalyzes the formation of the phosphoanhydride bond to generate ATP from ADP in the presence of phosphoryl source phosphocreatine (PCr). Herein, we employ these enzymes singularly and in tandem to work around with the adenosine-phosphate-induced self-assembly of NDPA.

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RESULTS AND DISCUSSION System Characteristics. The initial experiments were performed for the investigation of guest/fuel-induced selfassembly of NDPA by monitoring the changes in absorption

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DOI: 10.1021/jacs.7b07469 J. Am. Chem. Soc. 2017, 139, 16568−16575

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Journal of the American Chemical Society

diameter of ∼70 nm. These observations support that ATP, ADP, and AMP induce stronger interaction between chromophores, resulting in longer assemblies, and Pi forms shorter assemblies due to lack of additional interactions. Hence, a clear difference in size of supramolecular polymers formed by adenosine phosphates is observed in comparison to native state and Pi-induced state of NDPA. Similar conclusions are also evident from intensity percentage DLS data (Figure S3a). Corresponding circular dichroism (CD) spectra represent well-bisignated signals for adenosine-phosphate-induced selfassembly of NDPA (Figure 1c) with no interference of the CD signal by adenosine phosphates alone (Figure S3c). In the native state, achiral NDPA does not show any CD signal. ATP induces right-handed helical assembly, suggested by positive Cotton effects, whereas ADP and AMP induce left-handed helical assembly, suggested by negative Cotton effects. This induction of chirality is due to the presence of the chiral ribosesugar moiety in adenosine phosphates that biases the binding to the Zn−DPA site in NDPA.19 Due to lack of any chiral source, Pi does not induce any chirality to NDPA and thus shows zero CD signal and no Cotton effect. An important characteristic of this supramolecular assembly, which is also an essential criterion to obtain a transient system, is the dynamic and adaptive nature of the assembling monomers. To examine this, 5 × 10−5 M solution of (M)-NDPA-AMP assemblies were taken, and to it, aliquots of 0.25 equiv of ATP were added (Figure S3b). The solution initially showed a negative CD signal at 394 nm indicative of left-handed helical assembly. After each addition, an instantaneous increase from negative to positive CD signal is observed, which does not change over time. Addition of 1.0 equiv of ATP completely reverses the CD signal, suggesting a complete replacement of AMP from the NDPA assembly by the ATP-forming (P)-NDPA-ATP assembly. Thus, the system is highly adaptive and selective to the guest. Hence, NDPA forms fuel-selective right-handed helical (P)NDPA-ATP assemblies, left-handed helical (M)-NDPA-ADP and (M)-NDPA-AMP assemblies, and racemic (rac)-NDPA− Pi stacks (Scheme 1b, Figure 1a). Moreover, we realized a difference in the degree of polymerization of NDPA stacks which follows NDPA < NDPA−Pi < NDPA-AMP < NDPAADP ≈ NDPA-ATP.22 Additionally, these assemblies are highly dynamic and adaptive to the presence of fuel. This suggests that the rate of fuel transformation (by enzyme) directly determines the changes observed in NDPA organization and assembly. Hence, we envisage to temporally program the length and conformation of the NDPA assembly using fuels and enzymes. Single Nonequilibrium State Transient Self-Assembly. The system under study shows a fuel-selective, adaptive, and dynamic assembly. The next step is to create a transient selfassembly using adenosine phosphate fuels. In this study, we consider (M)-NDPA-ADP and (M)-NDPA-AMP states as a single nonequilibrium conformational state due to the lack of any clear spectroscopic signal to differentiate them, as both form left-handed helical stacks (Figure 1c). Thus, we considered left-handed and right-handed helical conformations as the two nonequilibrium conformational states. First, we investigated a simplistic single-state transient self-assembly using alkaline phosphatase (ALP) enzyme and ADP as fuel (Figure 2a, Figure S1). To attain this, 1.5 equiv of ADP is added to a solution of 5 × 10−5 M NDPA and 0.56 U/mL of ALP, and the resultant spectroscopic changes as a function of time are probed. Absorption changes monitored at the NDI

spectra (Figure 1a). The spectra were normalized at 362 nm to clearly elucidate the reversal of the vibronic bands of NDI

Figure 1. Fuel-dependent self-assembly of NDPA. (a) Absorption spectra normalized at 362 nm depicting extent of intermolecular interactions follows the order, (P)-NDPA-ATP > (M)-NDPA-ADP > (M)-NDPA-AMP > (rac)-NDPA−Pi. (b) Number-average DLS data showing that size of self-assembly follows the same order as in (a). (c) CD spectra representing helical organizations of assembly. ATP induces right-handed conformation, ADP and AMP induce left-handed conformation, and Pi does not induce chirality in NDPA assemblies. ([NDPA] = 5 × 10−5 M, 10 mM HEPES, [ATP] = 1 equiv, [ADP] = 1.5 equiv, [AMP] = 3 equiv, [Pi] = 3 equiv).

π−π* absorption, which is very sensitive to variation in regards to the extent of interchromophoric interactions. It is known that the lower intensity of the band at 383 nm with respect to band at 362 nm depicts the increasing extent of interchromophoric interactions. The intensity at 383 nm for NDPA (5 × 10−5 M, 10 mM HEPES) on interaction with various phosphates follows the order of NDPA > NDPA−Pi > NDPA-AMP > NDPA-ADP > NDPA-ATP where the ratio of the intensities of the two bands are 1.19, 1.06, 0.98, 0.93, and 0.91, respectively (Figure 1a). The values suggest that the reversal of the intensity of vibronic bands is induced by all three adenosine phosphate fuels ATP, ADP, and AMP, where the multivalent fuel interacts to form stronger assemblies.22 Although there is a change in vibronic features upon addition of Pi, no reversal of vibronic bands that describes a weak intermolecular interaction between the chromophores induced by Pi is observed. This can be well attributed to the fact that binding of Pi neutralizes the charge on Zn(II), which decreases the electrostatic repulsion between molecules; however, Pi lacks in additional hydrophobic, hydrogen bonding, and π−π interactions provided by the adenine base and ribose sugar in adenosine phosphate guests, along with the clipping of chromophores by multivalent guests ADP and ATP. To quantify the sizes of the supramolecular assemblies of adenosine-phosphate-bound assemblies, dynamic light-scattering (DLS) measurements were performed (Figure 1b). The number-average size data depict the longer assemblies for NDPA-ATP and NDPA-ADP (∼300 nm), with a slightly lower size of ∼200 nm for NDPA-AMP, whereas NDPA−Pi forms stacks of ∼100 nm, very close to the NDPA hydrodynamic 16570

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Figure 2. Transient self-assembly of NDPA with one conformational transient state fueled by ADP and mediated by alkaline phosphatase. (a) Schematic representation of transient cycle fueled by ADP. Dashed arrows represent refueling of the system. (b) Time-dependent absorption changes at λ = 394 nm representing transient assembly and its refueling with subsequent addition of ADP with enzyme-dependent extent of assembly and lifetime of nonequilibrium state. (c) Corresponding CD changes which shows the formation and decay of helical assembly. (d) Number percentage DLS size changes as a function of time at ALP = 1.12 U/mL, showing an instantaneous increase on addition of ADP and then decrease in size as cycle finishes ([NDPA] = 5 × 10−5 M, 10 mM HEPES, [ADP] = 1.5 equiv, [ALP] = 0.56, 1.12, and 1.68 U/mL). Arrows in (b) and (c) shows the (re)fueling points with the addition of ADP.

presence of a higher amount of enzyme as depicted by the CD signal, suggesting that the rate of disassembly as well as the fuel (ADP) consumption is faster (Figure S6). Similarly, we also attempted to fuel the system using AMP and employ alkaline phosphatase to mediate the transient assembly (Figure S7), which follows a similar trend. Thus, we demonstrated AMP/ADP-fueled single nonequilibrium state transient self-assembly mediated by ALP. Multistate, Nonequilibrium Transient Self-Assembly. After obtaining transient self-assembly with a single nonequilibrium conformational state, we opted for fueling the system with ATP. An in situ hydrolysis of ATP → ADP → AMP → Pi should reflect in chiral reorganization from the right-handed helical state to the left-handed helical state upon hydrolysis of ATP to AMP. However, further hydrolysis to Pi should result in disassembly as well as racemization as suggested by DLS of the fuel-adaptive NDPA assembly (Figure 1b,c). For this, 1 equiv of ATP is added to a solution of 5 × 10−5 M NDPA and ALP and then spectroscopic changes as a function of time are followed. Absorption changes at 394 nm show an instantaneous formation of (P)-NDPA-ATP assembly that undergoes a steady decay to the disassembled (rac)-NDPA−Pi state (Figures 3a,b and S8). However, the CD signal initially showed a positive value depicting the formation of right-handed helical (P)-NDPA-ATP state, which then gradually changed to a negative value illustrating the P → M stereomutation (conformational switching) and, hence, signifying the formation of the (M)-NDPA-ADP and the (M)-NDPA-AMP

aggregation band at 394 nm depict an instantaneous increase due to formation of ADP-induced assembly (NDPA-ADP) followed by a gradual decrease owing to in situ hydrolysis of ADP (→ AMP) → Pi (Figure 2b). The corresponding CD changes show an instantaneous increase in negative CD signal at 394 nm due to formation of the (M)-NDPA-ADP assembly. The signal gradually decays to zero, signifying racemization of the (M)-NDPA-ADP assembly into the (rac)-NDPA−Pi assembly (Figure 2c). Moreover, the number percentage DLS changes over the cycle show an increase in size of the NDPA stacks from ∼100 to ∼400 nm on binding with ADP, followed by a decrease to ∼120 nm upon hydrolysis (Figure 2d). This verifies that the changes are due to transient helical selfassembly, and the same trend is observed in intensity percentage measurements (Figure S4a). Subsequent addition of ADP refueled the transient system which follows a similar trend in absorption and CD, depicting the refueling ability of the system (arrows in Figure 2b,c show the refueling steps with ADP). To modulate the lifetime of the transient state and rate of disassembly, we varied the enzyme units. The lifetime of the nonequilibrium left-handed helical conformational state in the presence of 0.56, 1.12, and 1.68 U/mL obtained was 2901 ± 49, 1127 ± 129, and 372 ± 28 s, respectively. The corresponding rates of disassembly were 2 ± 1.13 × 10−3 s−1, 4.99 ± 0.9 × 10−3 s−1, and 1.37 ± 0.07 × 10−2 s−1 (Figure S4b, Table S1). Thus, an increase in enzyme units resulted in an acceleration of the rate of disassembly and a decrease in the lifetimes of transient species (Figure S5). Interestingly, the extent of assembly formation was also decreased in the 16571

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Figure 3. Transient self-assembly of NDPA with two conformational transient states fueled by ATP and mediated by alkaline phosphatase. (a) Schematic representation of transient cycle showing the activity of ALP as k1 ≈ k2. Dashed arrows represent refueling of the system. Time-dependent changes monitored at λ = 394 nm. (b) Absorption representing transient self-assembly and (c) CD representing transient conformational switching and refueling with subsequent addition of ATP. (d) Corresponding time-dependent CD changes in log scale of time for clearer elucidation of the two transient states. (e) DLS size changes as a function of time, showing a continuous decrease in size in the presence of 1.12 U/mL of ALP. Colored curly arrows are added to guide the eyes. Black arrows in (c−e) represent addition of ATP. (f) Change in rate of disassembly and lifetime of transient state in the presence of different units of ALP. ([NDPA] = 5 × 10−5 M, 10 mM HEPES, [ATP] = 1 equiv, [ALP] = 0.56, 1.12, and 1.68 U/ mL).

assembly (Figure 3c,d). Successive hydrolysis to Pi resulted in significant disassembly into racemic stacks as suggested by the lack of CD signal. To further prove the change in size, DLS was probed as a function of time, and it shows a fast increase in size upon addition of ATP, which gradually decreases in size (Figure 3e). Thus, we demonstrated an ATP-fueled transient self-assembly that undergoes a P → M conformational switch before its complete disassembly. Hence, we present a unique example of transient self-assembly with two nonequilibrium conformational states. The transient cycle was refueled by subsequent addition of ATP at different enzyme concentrations as represented by the arrows in Figure 3b−e. The enzymatic assistance in mediating transient assembly was then exploited further to control the rate of disassembly and the corresponding lifetime of the transient states. ALP concentrations of 0.56, 1.12, and 1.68 U/ mL resulted in a transient cycle lifetime of 3412, 1512 ± 104, and 696 ± 41 s, respectively (Figure 3f). We then deconvoluted the lifetime of right- and left-handed nonequilibrium conformational states (see Supporting Information, (SI)). In increasing order of enzyme units, the lifetime of the right-handed helical conformation (P)-NDPA-ATP was deduced to be 65, 82, and 47.5 s, respectively, whereas the lifetime of the left-handed helical conformation (M)-NDPA-ADP and (M)-NDPA-AMP together was 3347, 1430, and 649 s, respectively (Table S1). This suggests a gradual decrease in lifetime of both of the species with an increase in enzyme units. Although a highly modular system with two transient conformational states was obtained using ALP, we then envisaged to understand the effect of enzyme variation. We observed that due to the highly dynamic nature of NDPA, the enzyme kinetics are directly responsible for the observed rates.

Thus, any change in enzyme from the unselective phosphatase ALP to the comparatively selective apyrase (AP) should change the lifetime of the transient species. Commercially obtained AP contained an ATPase/ADPase ratio of 10:1, which proposed a rate of ATP hydrolysis ten times faster than that of ADP hydrolysis (Figure S2). We envisage that employing AP should result in enhancement in the lifetime of ADP by compensation of the lifetime of ATP, which should reflect in the corresponding NDPA assembly (Figure 4a). For this, the absorbance and CD changes at 394 nm for a solution of 5 × 10−5 M NDPA and 4.8 U/mL of AP are monitored as a function of time after the addition of ATP (Figures4b,c and S9). The rate of disassembly was calculated from the absorption trace, and the rate of the two stereomutations P → M and M → rac were calculated from CD traces (Figure 4b,c). The comparison of the rate of disassembly with the rate of stereomutations supports our hypothesis that the P → M rate is faster than the M → rac rate, and the latter stereomutation is the rate-determining step. Furthermore, comparison of the percentage lifetime of the (P)-NDPA-ATP state out of the complete lifetime of a transient cycle suggests that there is a decrease to 0.4% in the AP case in comparison to 6.3% in the ALP case (Table S1,S2). Thus, by the utilization of ATPselective enzyme, we have obtained selective enrichment in lifetime of the (M)-NDPA-ADP state by the compensation of lifetime of the (P)-NDPA-ATP state. An approximate 16 times decrement in the lifetime of the (P)-NDPA-ATP state is observed. The variation in AP units to 9.6 U/mL clearly elucidated the decrement in the lifetime of the (P)-NDPA-ATP state, which in this case is just 2 s. Further, refueling of the transient self-assembly, mediated by 9.6 U/mL of AP, shows no 16572

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reversible conformational switching recently shown by our group.18a The aim was to achieve a transient self-assembly that undergoes two reversible conformational switching steps via M → P → M in the assembled state en route to its disassembly. Such a complex network requires multiple pathways working simultaneously at different rates. For this, the bioinspired “enzyme in tandem” approach was employed where the two opposing enzymes ALP and CPK are used in one-pot. Since ALP is unselective toward phosphoanhydride bond hydrolysis and CPK is selective to the formation of ATP from ADP in the presence of phosphocreatine (PCr), we envisage to obtain a complex network of interconnected loops (Figure 5a,b). We began with a 5 × 10−5 M solution of NDPA with 7 U/ mL of CPK and 0.3 U/mL of ALP. To it, 5 equiv of PCr and 2 equiv of ADP were added (Figure 5c). An instantaneous formation of the (M)-NDPA-ADP assembly occurred, which is represented by negative CD signal. With time, an evolution of positive CD signal was observed corresponding to the formation of the (P)-NDPA-ATP assembly. This is due to the enzymatic formation of ATP from ADP by CPK in the presence of PCr. Then, a gradual decay back to (M)-NDPAADP, which disassembles significantly to (rac)-NDPA−Pi via the (M)-NDPA-AMP state, is observed. Hence, for the first time, we report a transient self-assembly with three nonequilibrium conformational states (Figure 5b). The system was also successfully refueled by subsequent addition of ADP, as represented by arrows (Figure 5c). Herein, PCr only acts as a phosphate donor for ATP formation, and any direct interference of PCr with NDPA assembly is not observed (Figure S12). Although, for proof-of-concept, we could achieve multistate transient self-assembly, the extent of the CD signals that depict the extents of formation of different conformational states were significantly different. Moreover, the second nonequilibrium left-handed helical state reached only an extent of −2.68 compared to the initial state of −7.53 mdeg (Figure 5b). We then attempted a similar experiment as stated above, with replacement of ALP with AP. We believe that the difference in selectivity between ALP and AP should change the lifetime of the transient species. Moreover, the difference in the rate constants of ATP and ADP hydrolysis, as observed in the previous case, should increase the lifetime of second Mconformational state. Furthermore, the lower rate of ADP hydrolysis via AP should also enhance the extent of the second M-conformational state. In order to achieve this scenario, to a solution of 5 × 10−5 M NDPA, 7 U/mL of CPK, 0.3 U/mL of AP, 5 equiv of PCr, and 3 equiv of ADP were added, and CD was monitored. As expected, the CD signal initially responded as negative, corresponding to the formation of the (M)-NDPAADP assembly. Then a gradual evolution of positive CD signal that signified formation of the (P)-NDPA-ATP assembly was observed, which subsequently transformed back to a negative CD signal, signifying the formation of the M-type assembly (Figure 5d). Finally, this signal further decayed to zero, depicting the disassembly/racemization. Interestingly, as envisaged, a near high extent of CD signal of the three conformational transient states were obtained. This can be attributed to the fact that changing the enzyme from ALP to AP decreases the rate of hydrolysis of ADP, and thus, a higher extent of CD signal is observed. We then attempted to modulate the signal further by increasing the AP units from 0.3 to 0.6 U/mL while keeping other components of the system same (Figure 5d). This, resulted in acceleration of ATP

Figure 4. Transient self-assembly fueled by ATP and mediated by apyrase (AP). (a) Schematic representation of transient cycle showing the activity of AP with k1 > k2. Dashed arrows represent refueling of the system. Time-dependent changes at 394 nm in the presence of different units of AP along with corresponding first-order kinetic rate constants and lifetime: (b) absorption and (c) CD changes. Arrows in (b) and (c) represent addition of ATP. ([NDPA] = 5 × 10−5 M, 10 mM HEPES, [ATP] = 1 equiv, [AP] = 4.8 and 9.6 U/mL).

sign of the (P)-NDPA-ATP state. This is due to very high rates of ATP hydrolysis that we are unable to capture in the instrument (Figure S10). The high difference in rates of ATP vs ADP hydrolysis via AP was further exploited to study the effect of perturbing the transient cycle by addition of fuel in midst of the cycle. We thought of using a lower enzyme concentration, i.e., 4.8 U/mL of AP, to decelerate the ADP hydrolysis step and obtain an appreciable lifetime and rate of ATP hydrolysis that can be well monitored. In such a system, a steady state (M)-NDPA-ADP assembly is observed that further decays very slowly (Figure S11). We then attempted to perturb the transient state of the (M)-NDPA-ADP state by addition of ATP. Upon addition of ATP, we observe an instantaneous formation of (P)-NDPAATP, which decays into (M)-NDPA-ADP over time (Figure S11). Subsequent refueling underwent a similar behavior. This supports that our system is highly adaptive and gets activated when fueled by ATP. We then pursued the final goal of this study to have multinonequilibrium state transient self-assembly. Here, the nonequilibrium states should be connected nonunidirectionally via networks of chemical (fuel) transformations. We have already seen the effect of ATP hydrolysis by ALP and AP to result in a two-state transient self-assembly. We envisaged to couple the current transient self-assembly with the temporally controlled 16573

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Figure 5. Transient self-assembly with three nonequilibrium conformational states fueled by ADP and PCr and mediated by alkaline phosphatase or apyrase with creatine phosphokinase in tandem. (a) Schematic representation of transient cycle. Dashed arrows represent refueling of the system. Transient cycle by alkaline phosphatase: (b) Time-dependent CD spectral changes representing conformational switching 0 → M → P → M → 0 accompanied by self-assembly. (c) CD changes at λ = 394 nm as a function of time and then refueling with subsequent addition of ATP. (d) Comparison of the two enzymes ALP and AP showing increase in lifetime of M-state on compensation of P-state. Arrows in (b−d) represent addition of ATP. ([NDPA] = 5 × 10−5 M, 10 mM HEPES; for blue: [ADP] = 2 equiv, [PCr] = 5 equiv, 9 U/mL of CPK, 0.2 U/mL of ALP; for red: [ADP] = 3 equiv, [PCr] = 5 equiv, 7 U/mL of CPK, 0.3 U/mL of AP; for green: ([ADP] = 3 equiv, [PCr] = 5 equiv, 7 U/mL of CPK, 0.6 U/mL of AP).

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hydrolysis that decreased the lifetime and extent of the (P)NDPA-ATP state. Thus, to achieve multistate conformational control in transient self-assembly with system undergoing through the first loop and then the second as envisaged, we should have a higher rate of the formation of ATP than the hydrolysis of ATP. This is shown to follow in the concentrations limits above 7 U/mL of CPK, 5 equiv of PCr, 2 equiv of ADP, and below 0.6 U/mL of ALP and 0.3 U/mL of AP. Hence, we are able to selectively enrich the lifetime of desired transient species in comparison to other transient states (Table S3).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b07469. Synthesis, methods, and supporting data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected] ORCID

Subi J. George: 0000-0003-4429-5237 Notes

CONCLUSION

The authors declare no competing financial interest.

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Herein, we have successfully demonstrated the programming of an NDPA assembly for up to three nonequilibrium conformational states during transient self-assembly with varying extents of changes and lifetimes by combinations of fuels and enzymes (Table S4). In conclusion, the work presented here suggests our system to be highly modular and adaptive toward different enzymes. We believe that future lifelike materials which urge for an active and autonomous behavior can be built using bioinspired strategies which involve biofuels, noncovalent interactions, high adaptiveness, and dynamics. This further approach could be to make nonequilibrium steady state and autonomous oscillating systems using dialysis membranes and continuous flow reactors.20 Additionally, material applications such as chiroptical technological systems with temporal control have not been investigated so far. Thus, we believe that the present study should bring these transient systems under limelight for future out-of-equilibrium materials with modular temporal control over their functions.

ACKNOWLEDGMENTS We thank Prof. C. N. R. Rao, FRS for his support and guidance. We would also like to thank JNCASR and the Department of Science and Technology, Government of India, for financial support. In addition, the funding from Sheiq Saqr Laboratory (SSL), JNCASR is also acknowledged.

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