Catalasomes: Switchable, Programmable Catalytic Nanoreactors

Lead Research Organisation: University of Bristol
Department Name: Chemistry

Abstract

The synthesis of organic molecules lies at the heart of the production of many of the materials that we rely on in the modern world, from pharmaceuticals through to fine chemicals and advanced materials. However, the process of synthesis, employing a 'toolbox' of techniques, applied in a tried and-tested order has not changed radically for many decades: we have added many tools, but the toolbox itself remains essentially the same as it was in first half of the twentieth century. A fundamental drawback with this approach is the time involved in designing, testing and delivering a new synthesis of a complex target, which can take from take months to years to achieve. How can nanotechnology be used to fundamentally change this?

Future gazing allows one to envisage a new way of producing complex molecules using small, hypothetical 'Universal Molecular Synthesisers' (UMS) that iterate rapidly to the best synthetic approach via evolutionary algorithms coupled with predictive modelling and feedback from real-time reaction analysis. Clearly the delivery of this 'disruptive technology' lies some considerable way in the future, but we can at least ask: how do we take the first steps towards this goal? More specifically: what would be the key functional components of a UMS?

We believe a prime candidate for investigation is a synthetic construct that fuses inorganic and biological components to produce a switchable, programmable catalytic nanoreactor: the "catalasome"; effectively a synthetic organelle. In this short proof-of-principle study we aim deliver functioning examples of catalasomes and show that the eventual product from a given reaction can be determined not by the reagents present or their order of addition, as would be the case in classic synthesis, but rather by the programming of a multiple-catalasome containing system.

Planned Impact

As this is a short, proof of concept project, immediate key beneficiaries (1-3 years) will be scientists working in the fields of nanotechnology, catalysis and synthesis. We anticipate that successful proof-of-principle will engender considerable new research activity at the intersection of these three fields, possibly creating an entirely new sub-discipline and accordingly we will target the communication of the research outputs to these key audiences. Subsequently, as the on-going programme matures, the audiences will expand to encompass areas of programming and complex systems control.

In the longer term (5 years+) the new programmable synthetic nanoreactors will be highly commercially attractive. We anticipate either licensing technology to specialist nanotech companies or establishing our own spin-out company. In the very long-term, providing developmental follow up studies are successful, we believe that catalasomes could be a genuine 'disruptive technology'. As is the case with all paradigm shifts, it is impossible to prejudge both the reach and significance of the potential impacts with any real confidence, but they could be huge with major opportunities for wealth creation and UK-based economic growth.

The ultimate goal of this on-going research programme is to deliver the 'Universal Molecular Synthesisers'. In the far future (30-50 years?) such systems may render many larger-scale industrial processes obsolete, instead synthesis would become a local, sustainable cottage industry. Taking pharmaceuticals as an example, (hypothetical) automatic personal diagnostic units could programme the UMS which would produce tailor-made drugs with no external intervention: this would be the ultimate in 'personalised medicine' which would impact profoundly on both health and society.

Beyond inspiring new research horizons, and delivering new disruptive technologies, we believe this project captures the imagination in a truly exciting manner, making it ideally suitable for public engagement activities in the near future.

Publications

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Description In this short, proof-of-principle project we set out to construct a 'catalasome' - a functional, switchable catalytic nanoreactor. The design concept of the catalasome is a mesoporous silica core, loaded with the desired functional catalyst surrounded by a lipid bilayer envelope that isolates the catalyst from the reaction media. The lipid bilayer contains a modified pore-forming membrane protein that acts as an on/off switch in response to an applied stimulus (light/chemical/pH etc), by opening and closing of the pore.

We have successfully produced a range of catalysts supported on appropriate mesoporous silicas, with varying morphologies: large and small pore nano and microparticles. In particular we have produce new tethered Grubbs-type catalysts, immobilised in the pores of nano- and microparticles that act as excellent heterogeneous catalysts for the metathesis of water-soluble substrates in aqueous reaction media and we are currently preparing a manuscript for publication.

We undertook extensive investigation of the encapsulation of spherical, catalyst-modified, large and small pore micro- and nanoparticles in lipid bilayers. This study focussed on identifying the ideal lipid composition, coupled with the ideal particle/pore size in order to be able to fully isolate the catalytic centres from the reaction media, whilst still being capable of supporting functional membrane proteins. With the idealised systems in hand we were able to obtain very good levels of catalyst isolation from the reaction media, with the ratio of rates of catalysis in the presence or absence of the lipid bilayer, with the idealised supported catalysts of ~20:1. Interestingly, this was strongly pH dependent. Importantly, removal of the lipid bilayer restored catalytic activity, indicating that the catalytic centres were truly isolated rather than degraded.

With the catalyst-functionalised core and supported lipid bilayer of representative catalasomes in hand, we next turned our attention to switching the catalasomes 'on' and 'off' by the incorporation of appropriate gates into the lipid bilayer. As described in the proposal, our efforts centred on the incorporation of a co-valently tagged MscL protein. The mutant MscL that we produced displayed good functional activity in test liposomes, in the presence of MTSET (an irreversible chemical activator). Unfortunately, while we were able to co-valently modify the mutant MscL with a spiropyran photochemical activator, despite very considerable efforts we were unable to reproduce that activity reported in the literature on photochemical activation of the gate and so far have not been able to produce a reversible photochemical gate and this remains an ongoing issue. Therefore we focussed on (irreversible) chemical switching in order to test the incorporation of functional MscL into the lipid bilayer of the catalasome.

It became apparent that MscL does not function in the lipid bilayers surrounding the silica cores. It seems likely that this is due to unfavourable interactions of the bulky C-helix at the base of the protein, which protrudes inside the liposome, with the silica surface. Accordingly we began to investigate ways of incorporating an aqueous 'buffer' layer between the silica surface and the lipid bilayer. Of the various approaches we have investigated so far, the most promising is the tethering of membrane proteins to the surface of the silica with long-chain bifunctional linkers. These consist of a silica tagging group at one end for surface immobilisation and a nitrilotriacetic acid (NTA) group which forms a complex with Ni(II) at the other. The Ni complex binds to the His tag at the C-terminus of the protein, anchoring it to the surface. For expedient spectroscopic characterisation, most of the scoping studies where performed with wild-type reaction centres from the purple photosynthetic bacterium Rhodobacter sphaeroides. Having established that the proteins could indeed be immobilised with the tether, preliminary investigations showed that MscL could also be tethered, that lipid bilayers could subsequently be formed and tantalisingly, using a calcein leak assay, we obtained preliminary data that suggests that at least some of the MscL remains functional after this protocol. Unfortunately, during the rather lengthy assembly procedure, the catalyst lost activity so we were unable to demonstrate whether or not catalyst switching could be achieved.

Alongside the protein gating studies, we investigated an alternative switching protocol, by introducing azobenzene amphiphiles into the lipid bilayer of a catalasome. The trans-form of the amphiphile leads to good packing in the lipid-bilayer, while the cis-form obtained on UV irradiation leads to a disruption in packing and membrane permeability. Unfortunately it transpired that the cis-form of the azobenzene acts as a poison for the modified Grubbs catalyst, a result replicated under homogeneous conditions.

In summary, during this short proof-of-principle study, we were able to construct both the functional catalytic core of the catalasome and the lipid bilayer envelop to surround it. The envelop does indeed lead to effective isolation of the core from the reaction media. The introduction of functional gates into the lipid bilayer has proven to be challenging, with significant issues occurring on interaction of the membrane protein with the silica surface. Future approaches will target (a) replacing the silica with a softer, more biocompatible core or (b) introducing a permeable soft layer between the core and the lipid bilayer and (c) moving to more robust, artificial gates.
Exploitation Route Plans for future investigation are briefly outlined in the key findings section
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology