The following functions for the metabolism are

  1. The ability to convert percursor molecules into the corresponding building blocks (amphiphiles and nucleic acids, i.e. the container and information building blocks)
  2. The ability to use a primary source of energy to achieve this conversion

Metabolism is often defined as the set of chemical reactions that occur in living organisms in order to maintain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments.

In other words, an important aspect of Living systems is their ability to convert primary forms of energy (chemical, light or heat energy) into usable forms of chemical energy that can be then used to produce their building blocks.

In the case of a protocell, the metabolism should be reduced to its most simple iteration: one single reaction that would produce or allows the synthesis of at least some of the protocell building blocks from precursor molecules present in the protocell environment.

The ability of nucleic acids to shuffle electrons along their double stranded structures allows one to consider a metabolism that is centered on electron transfer reactions. The cornerstone of such a metabolic process can then be a metal complex that excited by light irradiation can cleave a carbon-carbon bond on an amphiphile building block precursor under information control.

We (collaborative effort with the protocell team at LANL) have settled on a ruthenium trisbipyridine,
[Ru(II)(bpy)3]2+ (see Scheme 1 and Figure 1) because it permits us to introduce information control on the reaction. (The thermodynamics of this central catalytic reaction are quite well understood.7) It is well known that visible photoexcitation of [Ru(II)(bpy)3]2+ generates a metal-to-ligand charge transfer (MLCT) state that is both a better oxidant and reductant than the ground state.7 This excited state has been usedby many researchers to initiate electron transfer reactions.8 Our system is however designed by choosing a picolylester amphiphile precursor with a Ered = - 0.82 V to proceed via a reductive quenching pathway from the excited state (the left side of the diagram in Fig. 1, top) and proceeds only if the donor possesses an oxidation potential less negative than
~ -1.0 V. This represents an opportunity for information control. Indeed, an electron must be provided by the information molecule generating the reactive species [Ru(II)(bpy0)2(bpy-1)]1+ before the cleavage reaction of the amphiphile precursor can proceed (Fig. 1, Reaction 1). Once generated, the Ru1+ complex can then transfer charge to the picolinium ester providing carboxylic acid in the presence of a hydrogen source (Fig. 1, Reaction 2).

In a first design of the whole chemical system, we have simplified it by replacing the nucleic acid polymers by a single nucleobase and checked whether fatty acid amphiphiles can be generated from precursor molecules by visible light photolysis under single nucleobase control (Scheme 1).

Scheme 1. Simplified minimal chemical system. Decanoic picolylester (1), dihydrophenylglycine (2), 8-oxoguanine (3a), ruthenium catalyst (3b) and decanoic acid (4). In blue highlight are the container precursors and building blocks, in red the catalytic center and in green the simplified version of the information.

We have shown that the photo-induced electron transfer on the ruthenium complex could drive reductive cleavage of the amphiphile picolylester (1) to produce bilayer-forming molecules (4); specifically, visible photolysis in a mixture of a decanoic acid ester precursor, hydrogen donor molecules (2), and a ruthenium-based photocatalyst (3) that employs a linked nucleobase (8-oxo-guanine, 3a) as an electron donor, generates decanoic acid (Scheme 1 and Fig. 1, 4).5,6 (The 8-oxo-G is attached to one of the bipyridine ligands via an electronically insulating alkane bridge to avoid significant changes in the redox potential from that of the parent [Ru(II)(bpy)3]2+ complex).

Futhermore, guanine, the most easily oxidized conventional nucleobase, is not a sufficiently strong electron donor to provide an electron to the Ru MLCT excited state (see kinetics measurements in Figure 2).

Figure 1. A summary of the reductive and oxidative pathways for the [Ru(II)(bpy)3]2+ complex showing the redox potentials and reactions of the photocatalyzed ester cleavage reaction. Only the reductive quenching pathway is thermodynamically feasible. All electrode potentials are relative to N.H.E. from Vlcek et al.7

Figure 2. Kinetics of the reaction of decanoate precursor in the presence of 8-oxo-G-Ru catalyst (squares) or G-Ru complex (diamonds). Conversion is calculated using the loss of the absorbance due to the ester carbonyl stretching vibration at 1740 cm-1, an area free of interfering absorbance changes. There is no reaction beyond background with the guanine Ru complex. Labels A-F correspond to the same conversion levels shown in the images of Fig. 3, but occur at different times due to changes in the experimental set-up (light intensities, wavelengths and pathlengths). Initial reaction rates were estimated from the slope of a straight line fit to the 8-oxo-G curve for conversions of less than 40% vs. that to the guanine curve for times less than 400 minutes.
















Figure 3. Epifluorescence micrographs of aggregate formation mediated by the photolysis reaction. All reaction samples are stained with 2.5 µM Nile Red. The epifluorescence micrographs were taken under UV-illumination and observed through a 550-560 nm filter. Frames A) - D) are for the 8-oxo-G linked photocatalyst. A) at t = 0 h with 0% conversion. B) after 6-h irradiation with ~44% conversion, oil-water emulsion structures likely coated by fatty acid are freely moving in the medium. C) after 8-h irradiation with ~47% conversion, both emulsion and membranous structures are observed. Note the apparent shedding of material from the emulsion structures. D) after ~65% conversion at 24-h. Vesicles and other membranous structures are already readily apparent at ~50% conversion (not shown). Frames E) and F) are for the guanine analog of the photocatalyst. E) at t = 0 h with 0% conversion. F) at t= 24h. The bright spot visible is some of the fatty acid precursor that has "phase-separated" from solution.

In order for this chemical system to exhibit more of the necessary functions of a self-replicating network, several further steps will clearly be necessary.nAll the species must be co-located into/onto the container, the 8-oxo-G molecule must be incorporated into an oligomer or polymer with a base sequence that has the potential for replication, and the system must continue to function under these conditions. Our ongoing research efforts are striving to address these issues.