Adenosine-Phosphate-Fueled, Temporally Programmed

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Adenosine Phosphate-Fuelled, Temporally Programmed Supramolecular Polymers with Multiple Transient States Shikha Dhiman, Ankit Jain, Mohit Kumar, and Subi J. George J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07469 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017

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Journal of the American Chemical Society

Adenosine Phosphate-Fuelled, 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 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 fuel dependent helical assembly of a supramolecular polymer. We further attempt for 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 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.

Introduction Omnipresent inspirations for supramolecular chemistry are the naturally occurring systems.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 yet as an intriguing challenge.2 There have been attempts to obtain kinetically trapped assemblies that convert to more stable thermodynamic assemblies with time, that are highly dependent on the pathway of preparation.3 Although, the conversion of these kinetically trapped/metastable assemblies to thermodynamic assemblies occur over a time period, but 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 the future non-equilibrium 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 nonNaturally occurring self-assembled equilibrium.5,6 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-of-equilibrium 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 Taking inspiration from natural systems to create materials with spatio-temporal 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 α-chymotrypsin enzyme to form and disintegrate the small peptide assembling molecule, thereby presenting a biocatalytic12 transient gelation.13 Walther’s group reported a unique non-linear temporal control over pH change using urease enzyme. This urease enzyme can be coupled to any pH sensitive system to transiently modulate its activity. They further employed this temporal control over pH change to transiently program gelation of molecules, conformation of i-motif DNA and control photonics behavior of polymer.14

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Scheme 1. (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 multi-state helical conformational changes. (c) Graphical representation of transient self-assembly with multi non-equilibrium conformational state depicted in growth and conformation vs. time. In schematic (b) and (c), peach background represents right-handed (P) helical assembly, blue represents left-handed (M) helical assembly, grey represents disassembled/racemic state. Arrows in (b) and (c) indicate the addition or transformation of various fuels. The figure has NDPA as monomer in initial state, however, we are uncertain about its extent of assembly and racemic or achiral structure and thus, keep it as NDPA monomers. (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).

In a very interesting bioinspired approach, Prins and coworkers employed ATP, the biofuel, to drive the formation of vesicular nanoreactor in 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 zinc receptor functionalised 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 coworkers 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 towards 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, so is the case of 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 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 interconnected network of non-equilibrium 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 one-dimensional fibers on interaction with adenosine phosphate fuels. We have shown that these assemblies undergo adenosine phosphate 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 the requirement. 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 variety of fuel transformations via different pathways, as a consequence of which self-assembly of NDPA will undergo transformations (Scheme 1a-c), thus, making our system unique and modular. The enzyme used here are

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alkaline phosphatase (ALP), apyrase (AP) and creatine phosphokinase (CPK) (Scheme 1d-f, Figure S1-S2). Alkaline phosphatase hydrolyses the phosphoanhydride bond unselectively, whereas apyrase has a 10:1 ATPase to ADPase ratio that result in higher rate of ATP hydrolysis than ADP. In contrast, creatine phosphokinase catalyses the formation of phosphoanhydride bond to generate ATP from ADP in 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.

Results and Discussion System characteristics

ratio of intensity 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 intensity of vibronic bands is induced by all three adenosine phosphate fuels ATP, ADP and AMP where the multivalent fuel interact to form stronger assemblies. 22 Although, there is change in vibronic features on addition of Pi, but no reversal of vibronic bands is observed that describes a weak intermolecular interaction between the chromophores induced by Pi. This can be well attributed that binding of Pi neutralizes the charge on Zn(II) that decreases the electrostatic repulsion between molecules, however, Pi lacks in additional hydrophobic, hydrogen bonding and π-π interactions provided by adenine base and ribose sugar in adenosine phosphate guests, along with 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 NDPAADP (~300 nm), with slightly lower size of ~200 nm for NDPA-AMP, whereas NDPA-Pi forms stacks of ~100 nm, very close to NDPA hydrodynamic diameter of ~70 nm. These observations support that ATP, ADP and AMP induce stronger interaction between chromophores resulting in longer assemblies and Pi form 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).

Figure 1: Fuel-dependent self-assembly of NDPA. (a) Absorption spectra normalized at 362 nm depicting extent of intermolecular interactions follows the order, (P)-NDPAATP>(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 & AMP induce left-handed conformation and Pi doesn’t induce chirality in NDPA assemblies. ([NDPA] = 5x10-5 M, 10 mM HEPES, [ATP] = 1 eq., [ADP] = 1.5 eq., [AMP] = 3 eq., [Pi] = 3 eq.)

The initial experiments were performed for the investigation of guest/fuel induced self-assembly of NDPA by monitoring the changes in absorption spectra (Figure 1a). The spectra was normalized at 362 nm to clearly elucidate the reversal of vibronic bands of NDI π-π* absorption, which is very sensitive to variation with the extent of inter-chromophoric interactions. It is known that the lower intensity of band at 383 nm with respect to band at 362 nm depict increasing extent of interchromophoric interactions. The intensity at 383 nm for NDPA (5x10-5 M, 10 mM HEPES) on interaction with various phosphates follows the order of NDPA> NDPAPi> NDPA-AMP> NDPA-ADP> NDPA-ATP where the

Corresponding circular dichroism (CD) spectra represent well bisignated signals for adenosine phosphates induced self-assembly of NDPA (Figure 1c) with no interference of 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 effect whereas ADP and AMP induce left- handed helical assembly suggested by negative cotton effects. This induction of chirality is due to the presence of chiral ribose sugar moiety in adenosine phosphates that biases the binding to the ZnDPA site in NDPA.19 Due to lack of any chiral source, Pi do not induce any chirality to NDPA and thus show 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 dynamicity and adaptiveness of the assembling monomers. To examine this, 5x10-5 M solution of (M)-NDPA-AMP assemblies were taken and to it aliquots of 0.25 eq. 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 doesn’t change over time. Addition of 1.0 eq. ATP completely reverses the CD signal suggesting a complete



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replacement of AMP from NDPA assembly by ATP forming (P)-NDPA-ATP assembly. Thus, system is highly adaptive and selective to guest. Hence, NDPA forms fuel-selective right handed helical (P)-NDPA-ATP assembly, left handed helical (M)NDPA-ADP and (M)-NDPA-AMP assembly 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< NDPAAMP< NDPA-ADP~ NDPA-ATP.22 Additionally, these assemblies are highly dynamic and adaptive to the presence of fuel. This suggests that rate of fuel transformation (by enzyme) directly determine the changes observed in NDPA organization and assembly. Hence we envisage to temporally program the length and conformation of NDPA assembly using fuels and enzymes. Single non-equilibrium state transient self-assembly

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 arrow 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] = 5x10-5 M, 10 mM HEPES, [ADP] = 1.5 eq., [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.

The system under study shows a fuel-selective, adaptive and dynamic assembly. The next step is to create a transient self-assembly using adenosine phosphate fuels. In this study, we consider (M)-NDPA-ADP and (M)-

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NDPA-AMP states as a single non-equilibrium conformational state due to 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 eq. ADP is added to a solution of 5x10-5 M NDPA and 0.56 U/mL ALP and the resultant spectroscopic changes as a function of time is probed. Absorption changes monitored at the NDI 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 (M)-NDPA-ADP assembly. The signal gradually decays to zero CD signal signifying racemization of (M)-NDPAADP assembly into (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 nm 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 refuel-ability of the system (arrows in Figure 2b and c shows the refueling steps with ADP). To modulate lifetime of transient state and rate of disassembly, we varied the enzyme units. Lifetime of non-equilibrium left-handed helical conformational state in presence of 0.56 U/mL, 1.12 U/mL and 1.68 U/mL obtained was 2901±49 seconds, 1127±129 seconds and 372±28 seconds, respectively. The corresponding rates of disassembly were 2±1.13x10-3 s-1, 4.99±0.9x10-3 s-1 and 1.37±0.07x10-2 s-1 (Figure S4b, Table S1). Thus, increase in enzyme units resulted in acceleration of rate of disassembly and decrease in lifetime of transient species (Figure S5). Interestingly, the extent of assembly formation was also decreased in presence of higher amount of enzyme as depicted by CD signal suggesting that 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 non-equilibrium state transient self-assembly mediated by ALP. Multi state, non-equilibrium transient self-assembly After obtaining transient self-assembly with a single nonequilibrium conformational state, we opted for fueling the system with ATP.


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Figure 3: Transient self-assembly of NDPA with 2 conformational transient states fueled by ATP mediated by Alkaline Phosphatase. (a) Schematic representation of transient cycle showing the activity of ALP as k1~k2. Dashed arrow 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 presence of 1.12 U/mL ALP. Colored curly arrows are put to guide the eyes. Black arrows in (c) to (e) represent addition of ATP. (f) Change in rate of disassembly and lifetime of transient state in presence of different units of ALP. ([NDPA] = 5x10-5 M, 10 mM HEPES, [ATP] = 1 eq., [ALP] = 0.56, 1.12 and 1.68 U/mL)

An in situ hydrolysis of ATP ADP AMP Pi should reflect in chiral reorganization from right-handed helical state to left-handed helical state on 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 eq. ATP is added to solution of 5x10-5 M NDPA and ALP, 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 undergo a steady decay to disassembled (rac)-NDPAPi state (Figure 3a,b and S8). However, CD signal initially showed a positive value depicting the formation of right handed helical, (P)-NDPA-ATP, state which then gradually change to negative value illustrating the P M stereomutation (conformational switching), hence signifying the formation of (M)-NDPA-ADP and (M)NDPA-AMP assembly (Figure 3c,d). Successive hydrolysis to Pi resulted in significant disassembly into racemic stacks suggested by zero CD signal. To further prove the change in size, DLS was probed as a function of time which shows a fast increase in size on addition of ATP that gradually decrease in size (Figure 3e). Thus, we demonstrated an ATP-fuelled transient self-assembly that undergo a P M

conformational switch before its complete disassembly. Hence, we present a unique example of transient selfassembly with two non-equilibrium 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 rate of disassembly and the corresponding lifetime of the transient states. ALP concentrations of 0.56 U/mL, 1.12 U/mL and 1.68 U/mL resulted in lifetime of transient cycle of 3412 seconds, 1512±104 seconds and 696±41 seconds respectively (Figure 3f). We then deconvoluted the lifetime of right- and lefthanded non-equilibrium conformational states (see SI). In increasing order of enzyme units the lifetime of right handed helical conformation (P)-NDPA-ATP were deduced to be 65, 82 and 47.5 seconds, whereas the lifetime of left handed helical conformation (M)-NDPA-ADP and (M)-NDPA-AMP together were 3347, 1430 and 649 seconds respectively (Table S1). This suggest a gradual decrease in lifetime of both the species with increase in enzyme units. Although a highly modular system with two transient conformational state was obtained using ALP, we then envisaged to understand the effect of enzyme



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variation. We observed that due to highly dynamic nature of NDPA, the enzyme kinetics are directly responsible for the observed rates. Thus, change in enzyme from unselective phosphatase ALP to comparatively selective Apyrase (AP) should change the lifetime of transient species. Commercially obtained AP constituted of ATPase:ADPase ratio of 10:1, proposing a ten times higher rate of ATP hydrolysis than ADP (Figure S2).

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of two stereomutations P M and M rac were calculated from CD traces (Figure 4b,c). The comparison of the rate of disassembly with rate of stereomutations support our hypothesis that P M rate is faster than M rac stereomutation and the later stereomutation is the rate determining step. Furthermore, comparison of percentage lifetime of (P)-NDPA-ATP state out of the complete lifetime of a transient cycle suggests that there is a decrease to 0.4% in AP case in comparison to 6.3% in ALP case (Table S1-S2). Thus, by the utilization of ATP selective enzyme, we have obtained selective enrichment in lifetime of (M)-NDPA-ADP state by the compensation of lifetime of (P)-NDPA-ATP state. Approximately 16 times decrement in lifetime of (P)NDPA-ATP state is observed. The variation in AP units to 9.6 U/mL clearly elucidated the decrement in lifetime of (P)-NDPA-ATP state, which in this case is just 2 seconds. Further, refueling of the transient self-assembly mediated by 9.6 U/mL AP, show no sign of (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 by 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 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 decay very slowly (Figure S11). We then attempted to perturb the transient state of (M)NDPA-ADP state by addition of ATP. On addition of ATP, we observe an instantaneous formation of (P)-NDPA-ATP 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 fuelled by 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 arrow represent refueling of the system. Time dependent changes at 394 nm in presence of different units of AP, (b) absorption, (c) CD changes and corresponding first order kinetic rate constants and lifetime. Arrows in (b) and (c) represent addition of ATP. ([NDPA] = 5x10-5 M, 10 mM HEPES, [ATP] = 1 eq., [AP] = 4.8 and 9.6 U/mL)

We envisage that employing AP should result in enhancement in lifetime of ADP by compensation of lifetime of ATP which should reflect in the corresponding NDPA assembly (Figure 4a). For this a solution of 5x10-5 M NDPA and 4.8 U/mL AP is taken and absorbance and CD changes at 394 nm is monitored as a function of time after addition of ATP (Figure 4b,c and S9). The rate of disassembly was calculated from absorption trace and rate

We then pursued the final goal of this study to have multi non-equilibrium state transient self-assembly. Here, the non-equilibrium states should be connected via networks of chemical (fuel) transformations nonunidirectionally. We have already seen the effect of ATP hydrolysis by ALP and AP to result in 2-state transient selfassembly. We envisaged to couple the current transient self-assembly with the temporally controlled reversible conformational switching shown by our group recently.18a The aim was to achieve a transient self-assembly that undergo two reversible conformational switching 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 bio-inspired “enzyme in tandem” approach was employed where the two opposing enzymes ALP and CPK are used in one-pot. Since ALP is unselective towards phosphoanhydride bond hydrolysis and CPK is selective to formation of ATP from ADP in presence of phosphocreatine (PCr), we envisage to obtain a complex network of inter connected loops (Figure 5a,b).


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Figure 5: Transient self-assembly with 3 non-equilibrium conformational states fueled by ADP and PCr mediated by Alkaline Phosphatase or Apyrase with Creatine phosphokinase in tandem. (a) Schematic representation of transient cycle. Dashed arrow 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 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) to (d) represent addition of ATP. ([NDPA] = 5x10-5 M, 10 mM HEPES; for blue: [ADP] = 2 eq., [PCr] = 5 eq., 9 U/mL CPK, 0.2 U/mL ALP; for red: [ADP] = 3 eq., [PCr] = 5 eq., 7 U/mL CPK, 0.3 U/mL AP; for green: ([ADP] = 3 eq., [PCr] = 5 eq., 7 U/mL CPK, 0.6 U/mL AP)

We began with a 5x10-5 M solution of NDPA with 7 U/mL of CPK and 0.3 U/mL of ALP. To it, 5 eq. PCr and 2 eq. of ADP were added (Figure 5c). An instantaneous formation of (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 (P)-NDPA-ATP assembly. This is due to the enzymatic formation of ATP from ADP by CPK in presence of PCr. Then a gradual decay back to (M)-NDPA-ADP is observed which disassembles significantly to (rac)-NDPAPi via (M)-NDPA-AMP state. Hence, for the first time we report a transient self-assembly with three nonequilibrium conformational state (Figure 5b). The system was also successfully refueled by subsequent addition of ADP represented by arrow (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 a proof-of-concept we could achieve multi-state transient self-assembly the extent of CD signals that depict the extents of formation of different conformational states were significantly different. Moreover, the second non-equilibrium left handed helical state reached only an extent of -2.68 compared to 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 transient species. Moreover the difference in 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 by AP should also enhance the extent of second M-conformational state. In order to achieve this scenario, to a solution of 5x10-5 M NDPA, 7 U/mL CPK, 0.3 U/mL AP and 5 eq. PCr, 3 eq. of ADP was added and CD was monitored. As expected, CD signal initially responded as negative corresponding to the formation of (M)-NDPAADP assembly. Then a gradual evolution of positive CD signal that signify formation of (P)-NDPA-ATP assembly was observed, which subsequently transformed back to a negative CD signal, that signifies the formation of M-type assembly (Figure 5d). Finally, this signal further decayed to zero CD signal depicting the disassembly/racemization. Interestingly, as envisaged, nearly 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 U/mL to 0.6 U/mL while keeping other components of the system same (Figure 5d). This, resulted in acceleration of ATP hydrolysis that decreased the lifetime and extent of (P)-NDPA-ATP state. Thus, to achieve multistate conformational control in transient selfassembly with system undergoing through the first loop and then second as envisaged, we should have higher rate of ATP formation than its hydrolysis. This is shown to follow in the concentrations limits above 7 U/mL of CPK, 5 eq. of PCr, 2 eq. of ADP and below 0.6 U/mL ALP and 0.3



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U/mL 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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Conclusion Herein, we have successfully demonstrated the programming of NDPA assembly for upto 3 nonequilibrium conformational state during transient selfassembly with varying extent of changes and lifetimes by combinations of fuels and enzymes (Table S4). In conclusion, the work presented here suggests our system to be a highly modular and adaptive towards different enzymes. We believe that future lifelike materials which urge for an active and autonomous behavior can be build using bio-inspired strategies which involve bio-fuels, noncovalent interactions, high adaptiveness and dynamics. The further approach could be to make non-equilibrium 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.

ASSOCIATED CONTENT Synthesis, methods and supporting data. This material is available free of charge via the Internet at http://pubs.acs.org.”

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AUTHOR INFORMATION Corresponding Author [email protected], [email protected]

ACKNOWLEDGMENT 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.

10.

ABBREVIATIONS

12.

11.

ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; Pi, inorganic phosphate; ALP, alkaline phosphatase; AP, apyrase; CD, circular dichroism; CPK, Creatine phosphokinase; PCr, Phosphocreatine.

13.

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