Characterisation of the system

Key to the operation of our system is a catalyst that is capable of both making and breaking new covalent bonds. We chose to use a ruthenium-catalysed alkene cross-metathesis approach for which, as far as we are aware, there is no known analogous reactivity in living systems. However, Ru-mediated metathesis is generally robust and versatile, and compatible with water28,29,30. Using hydrophilic alkene 1 and hydrophobic alkenes 2a–b, which are phase separated (Fig. 2), and Grubbs 2nd generation catalyst in D 2 O we studied the evolution of the different species generated with hydrophobic alkenes 2a (Fig. 2a) and 2b (Fig. 2b), which differ only by their carbon chain lengths. Both alkenes show similar behaviour, displaying sigmoidal kinetics characteristic of autocatalytic systems18,27. The initial lag period is much longer for hydrophobic alkene 2b, probably due to its reduced water miscibility (Fig. 2a vs. 2b). While consumption of hydrophilic alkene 1 shows classical sigmoidal autocatalytic behaviour (see blue line in Fig. 2b), the formation of amphiphilic 3 shows exponential growth with an initial lag period. After reaching a maximum point, 3 then gets completely consumed (see red line in Fig. 2b). The decomposition of replicator 3 gives soluble 4, which we believe is the thermodynamic product. Interestingly, formation of 4 does not occur from the beginning of the reaction, but only when a significant concentration of amphiphile 3 is reached and hydrophilic alkene 1 is almost consumed (see Fig. 2a, b). This would imply that only when the surfactant is self-assembled is it destroyed, in analogy to the disassembly of microtubules, in which it is the association of tubulin-GTP units in the supramolecular structure which activates the hydrolysis and eventual disassembly of the entire structure17,31.

Fig. 2 Kinetics of the self-replicating system showing concentration versus time of 1 (blue), 3 (red) and 4 (black). a The reaction of hydrophobic alkene 2a with a shorter carbon chain, shows autocatalytic kinetics in the consumption of 1 and formation of 3a. After reaching a maximum, thermodynamically unstable product 3a is consumed to form waste product 4. b The reaction of hydrophobic alkene 2b with a longer carbon chain again shows autocatalytic kinetics with a longer lag-period on account of the reduced water miscibility of alkene 2b. The peak in the concentration of 3b is again followed by its consumption to form waste product 4. c Re-fuelling starting materials 1 and 2a, once 3a is depleted, allows for the self-replication to resume. An increased stirring rate is used to increase the rate of the reaction between 1 and 2a, but the same pattern observed in Fig. 2a emerges. d Reaction of hydrophobic alkene 2b, seeding with 20 mol% of surfactant 3b. In this case waste product 4 is formed from the beginning as the metastable replictator is already present. Error bars represent standard deviation obtained from three repetitions Full size image

To demonstrate the potential for regeneration of the self-replicator, the reaction between 1 and 2a was run in batch, and then once replicator 3a had almost been fully consumed, starting materials 1 and 2a were resupplied (Fig. 2c). These experiments show that a population of replicator 3a can be regenerated before the inevitable conversion to product 4, and this process was repeated a third time without fatigue. Moreover, these experiments show the potential to control the self replicator population and maintain a far-from-equilibrium state of the metastable surfactant self-replicator.

The autocatalytic behaviour of 3b was further studied by performing seeding experiments, where addition of amphiphile 3b from the beginning of the reaction suppressed the initial lag-period in both consumption of starting material 1 and formation of cross-product 3b (Fig. 2d and Supplementary Fig. 14).

The aggregation properties of the amphiphiles 3a–b were studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM), and the critical micelle concentration (CMC) was determined using an established fluorimetric method (Fig. 3 and Supplementary Fig. 16–21).

Fig. 3 Characterisation of self-assembled amphiphile 3a. a DLS shows good correlation with an average size particle of 28 nm. b TEM image showing particles with an average diameter of 27 nm (scale bar of 200 nm). c Emission vs. concentration and CMC determination Full size image

Mechanistic experiments

In order to probe the reaction pathways that operate in this complex biphasic system, which will inform on the design of subsequent far-from-equilibrium self-replicators, and help us to better understand what features of the current system are necessary for its successful operation, we carried out a series of control experiments (Fig. 4). What emerges from the experiments described below is that, at least for the current system described here, the biphasic nature of the reaction medium, and a flow of energy from starting materials to products are both key for allowing concurrent formation and destruction steps, and, therefore, the far-from-equilibrium state.

Fig. 4 Mechanistic hypothesis supported by control experiments. a Diagram of the dynamic system including the chemical structures involved. Kinetic analysis representing concentration vs. time of 1 (blue), 3a (red) and 4 (black) for the control experiments: b In D 2 O replicator 3a is stable. It is only on addition of alkene 2a that it is fully consumed to form waste product 4. c In reaction between 1 and 2a performed under homogeneous conditions no autocatalytic kinetics are observed for the formation of 3a. Under these conditions product 3a appears to be relatively stable. d Qualitative energy profile of the system, highlighting the higher effective barrier for direct conversion of 1 to 4 under phase separated conditions. Phase separation may shift the equilibrium position between 3a and 4 by effectively removing water soluble 4 from the micellar environment Full size image

Mechanistically, we propose that reactions initiate in the organic phase (I—Phase separation in Fig. 4a), where the ruthenium catalyst activates the hydrophobic alkene 2, generating an active hydrophobic ruthenocarbene (II—Interfacial reaction in Fig. 4a). This then reacts with hydrophilic alkene 1 at the interface to give a ruthenocycle that collapses to amphiphilic 3. As the concentration of amphiphilic product 3 increases it self-assembles into micelles (III— Physical autocatalysis in Fig. 4a), and likely incorporates organic material into the micelles, as well as additional activated ruthenocarbenes generated from the decomposition of 3 (IV—Micelle destruction in Fig. 4a)30. The productive pathway towards the thermodynamic product 4 will therefore consume 3 (V—Thermodynamic product in Fig. 4a).

This mechanism is supported by the observation that hydrophilic alkene 1, in the presence of Grubbs 2nd generation catalyst, does not react in water (Supplementary Fig. 15a) to give dimeric 4, which is the thermodynamic waste product of our system. Amphiphile 3a in the presence of the metathesis catalyst in water also appears stable and does not react to form 4. However, if hydrophobic alkene 2a is added to 3a in water then it is fully consumed to give 4 (Fig. 4b). This experiment suggests that Grubbs 2nd may not be the catalytic active species responsible for destruction of 3, but that an activated ruthenocarbene derived from the hydrophobic alkene is involved.

A second set of control reactions was performed under homogeneous conditions using a t-BuOH-D 2 O solvent mixture, where all reaction components are soluble. Mixing 1, 2a, and catalyst under homogeneous conditions (Fig. 4c) showed rapid formation of 3a with no lag-period, and the reaction no longer appears autocatalytic. Homodimer 4 is formed in small quantities from the beginning, presumably from the dimerisation of 1, and the concentration of 4 then slightly increases with a concomitant reduction in the concentration of 3a (Fig. 4c). The selective and rapid formation of 3a compared to the slow dimerisation of 1 to form 4 under homogeneous condition is consistent with a study on cross-metathesis methods32,33. Formation of 4 from 1 was confirmed by a separate control experiment, in the absence of hydrophobic alkene 2a (Supplementary Fig. 15b). The incomplete conversion of 3a to 4 is also observed in a separate homogeneous control reaction (Supplementary Fig. 15c), which is in contrast to the relatively rapid conversion of 3a to 4 under biphasic conditions (see Figs. 2a or 4b).

While phase separation is a requirement for physical autocatalysis, why biphasic conditions are necessary for replicator destruction is less obvious, and both kinetic and thermodynamic factors may be at play. First, from the perspective of 3a, higher local concentrations of ruthenocarbenes in micelles of 3a would be expected than under homogeneous conditions, and this would facilitate the destruction of 3a. The effective equilibrium position may be altered between phase separated and homogenous conditions. For example, on destruction of 3a to form 4 and 2a, via 5, the phase separation of the extruded products might push the equilibrium towards increased consumption of 3a (Fig. 4). What is clear is that the phase separation both allows for micelle-mediated autocatalysis to occur and for the productive consumption of the self-replicator.

In conclusion, we have designed and operated a small molecule self-replicator whose formation is triggered by a physical autocatalytic reaction across a phase separation. In closed systems the maximum population of the self-assembled replicator is formed following a non-linear, far-from-equilibrium regime. The metastable self-assembled replicator is concurrently consumed, becomes depleted and moves towards thermodynamic equilibrium with the formation of a waste product. Addition of chemical fuel allows high replicator populations to be restored before the inevitable move toward equilibrium. Because formation and destruction of the self-replicator occur concurrently, the system is dynamic and allows temporal control of the self-replicator population. These studies demonstrate that for a small molecule self-replicator, a far-from-equilibrium population of replicators can be controlled in a fully synthetic system designed on first principles of chemical reactivity. Overall, the thermodynamic instability of the replicator enables the system to mimic two fundamental properties of living systems—the ability to self-replicate and persist far-from-equilibrium. Many living systems grosso modo are replicators working far-from-equilibrium, which are inevitably destroyed unless they are sustained. Study of dynamic metastable replicators may help to understand how to create minimal life in the laboratory, and provide physical models to study these fundamental properties outside of living systems.