Stefan Borsley

Biology routinely transduces energy stored in chemical bonds to create transmembrane gradients, which are themselves a crucial on-demand energy store to drive life’s vital processes. We report a strategy for the chemically fueled active transport of molecular cargo in large unilamellar vesicles. We outline the features required for driving active transport across phospholipid bilayers. Through a series of experiments in both hybrid lipid–liquid supported membranes and biomimetic vesicles, we demonstrate the transmembrane transport of our diacid cargo and quantify key performance indicators of our system. The active transport mechanism gives rise to an associated generation of transmembrane pH gradients of up to ∼0.3 pH units, comparable to the gradient required to drive ATP synthase. We harness this gradient as an energy source to drive a secondary active transport process through symport/antiport mechanisms. These chemically fueled active transport cascades demonstrate the successive transduction of free energy from chemical bonds to transmembrane proton gradients to transmembrane Na+ or Cl– gradients. Our work establishes the first example of chemically fueled active transport in unilamellar vesicles and demonstrates how transmembrane gradients can both absorb energy and transfer it as part of a greater systems-level, life-like, compartmentalized nanotechnology.

The structural anisotropy necessary to distinguish clockwise from counterclockwise motions in motor-molecules continuously rotating about a covalent single bond has previously been supplied by chiral fuelling systems or by enzymes. Here we report a class of rotary motors in which, like motor proteins, structural asymmetry in the motor itself causes directional rotary catalysis. A single stereogenic centre in azaindole–phenylethanoic acid motors is sufficient to produce diastereomeric intermediates of atropisomeric conformations in the catalytic cycle, generating 8:1 clockwise:counterclockwise directional bias in the motor’s rotary catalysis of diisopropylcarbodiimide hydration (motor substituent PhCH₂–). One enantiomer of a chiral hydrolysis promoter increases the directionality to 30:1 for clockwise rotation (motor substituent CH₃–), while the other enantiomer reverses the direction to 1:2 clockwise:counterclockwise. The experimental demonstration that a chiral molecular motor can be powered by a chemical fuel to rotate either with, or counter to, the motor’s dominant power stroke informs the understanding of how chemical energy is transduced through catalysis, the fundamental process that powers biology.

Denton's redox-neutral catalytic Mitsunobu reaction is remarkable in that it translates a reaction traditionally driven by the consumption of sacrificial chemical reagents to an additive-free catalytic manifold. Rational attempts to improve the system have been met with only marginal improvements, and a lack of consensus concerning the rate-determining step continues to limit effective reaction development. Here, we analyze the reaction mechanism focusing on a critical, largely overlooked element: the removal of water using a Dean-Stark apparatus. Experimental analysis of the water removal process, coupled with extensive kinetic simulations, demonstrates that the overall rate of the reaction is intimately tied to the rate of water removal. This process can be viewed as a transition between potential energy surfaces and, consequently, subsequent steps of the reaction can progress spontaneously in the absence of water, allowing an explanation of how Le Chatelier's principle, a thermodynamic effect, can have a profound kinetic influence over the rate of the reaction. We identify three bottlenecks in the reaction that inform catalyst design. Additionally, we (a) clarify the ongoing discussion regarding the rate-determining step, (b) provide clear advice concerning future reaction design taking into account the role of water and, (c) discuss the redox-neutral catalytic Mitsunobu reaction in the context of formally endergonic esterification reactions, noting parallels with ratchet mechanisms. Finally, we highlight general principles of catalyst/reaction design that emerge from our analysis and implement our findings to demonstrate a 50% rate acceleration resulting from improved water removal, a substantially greater reaction enhancement than previously obtained from computationally guided catalyst structural changes.

The structural anisotropy necessary to distinguish clockwise from counter-clockwise motions in motor-molecules that continuously rotate about a covalent single bond has previously been supplied through the use of chiral chemical fuels or with enzymes. Here we report on a new class of chiral azaindole–phenylethanoic acid rotary motors in which, like motor proteins, anisotropy is inherent through structural asymmetry in the motor itself. The motor-molecules consist of a 7-azaindole group attached through a C–N bond to a phenyl ring bearing a 2-(R or S)-(alkyl)ethanoic acid side chain. As the carboxylic acid group of the motor catalyses hydration of a carbodiimide fuel, the phenyl ring continuously directionally rotates about the azaindole stator. The stereogenic centre on the rotor side chain gives rise to diastereomeric conformations in both of the chemically distinct states (carboxylic acid and acyl-pyridinium) of the motor during the catalytic cycle. This generates a directional bias of 8:1 (clockwise:counter-clockwise; alkyl substituent = C6H5CH2-) for motor rotation powered by an achiral fuel, diisopropylcarbodiimide. The addition of one enantiomer of a hydrolysis promoter to the fuelling system increases the directionality of clockwise motor rotation to >30:1 (alkyl substituent = CH3-). The addition of the other enantiomer reverses the direction of catalysis-driven motor rotation (1:2 clockwise:counter-clockwise; alkyl substituent = CH3-). The experimental demonstration that a chiral molecular rotary motor can be powered by the same chemical fuel to rotate either with, or counter to, the motor’s dominant power stroke informs the understanding of how chemical energy is transduced through catalysis, the fundamental process that powers biology.

Cells display a range of mechanical activities generated by motor proteins powered through catalysis¹. This raises the fundamental question of how the acceleration of a chemical reaction can enable the energy released from that reaction to be transduced (and, consequently, work to be done) by a molecular catalyst^2–7. Here we demonstrate the molecular-level transduction of chemical energy to mechanical force⁸ in the form of the powered contraction and powered re-expansion of a cross-linked polymer gel driven by the directional rotation of artificial catalysis-driven⁹ molecular motors. Continuous 360° rotation of the rotor about the stator of the catalysis-driven motor-molecules incorporated in the polymeric framework of the gel twists the polymer chains of the cross-linked network around one another. This progressively increases writhe and tightens entanglements, causing a macroscopic contraction of the gel to approximately 70% of its original volume. The subsequent addition of the opposite enantiomer fuelling system powers the rotation of the motor-molecules in the reverse direction, unwinding the entanglements and causing the gel to re-expand. Continued powered twisting of the strands in the new direction causes the gel to re-contract. In addition to actuation, motor-molecule rotation in the gel produces other chemical and physical outcomes, including changes in the Young modulus and storage modulus—the latter is proportional to the increase in strand crossings resulting from motor rotation. The experimental demonstration of work against a load by a synthetic organocatalyst, and its mechanism of energy transduction⁶, informs both the debate^3,5,7 surrounding the mechanism of force generation by biological motors and the design principles^6,10–14 for artificial molecular nanotechnology.

Biology masters energy transduction, converting energy between various forms, and employing it to drive its vital processes. Central to this is the ability to use chemical energy for the active transport of substances, pumping ions and molecules across hydrophobic lipid membranes between aqueous (sub)cellular compartments. Biology employs information ratchet mechanisms, where kinetic asymmetry in the fuel-to-waste (i.e., substrate-to-product) conversion results in catalysis-driven active transport. Here, we report an artificial system for catalysis-driven active transport across a liquid hydrophobic membrane, pumping a maleic acid cargo between aqueous compartments. We employ two strategies to differentiate the conditions in either compartment, showing that transmembrane active transport can be driven either by adding fuel to a single compartment, or by differentiating the rates of activation and/or hydrolysis when fuel is present in both compartments. We characterize the nonequilibrium system through complete kinetic analysis. Finally, we quantify the energy transduction achieved by the catalysis-driven active transport, and establish the emergence of positive and negative feedback mechanisms within the system.

Cells display a range of mechanical activities enabled by the cytoskeleton, a viscoelastic hydrogel manipulated by motor proteins powered through catalysis. This raises the question of how the acceleration of a chemical reaction can enable the energy released from that reaction to be transduced, and thereby work to be done, by a molecular catalyst. Here we demonstrate the molecular-level transduction of chemical energy to mechanical force in the form of the powered contraction and powered re-expansion of a crosslinked polymer gel driven by the directional rotation of embedded artificial catalysis-driven molecular motors. Continuous 360° rotation of the rotor about the stator of motor-molecules incorporated within the polymeric framework of the gel, twists the polymer chains of the crosslinked network around one another (either clockwise or anti-clockwise, depending on the chirality of the fuelling system). This progressively increases writhe and tightens entanglements, causing macroscopic contraction of the gel to ~70% of its original volume. The limit of contraction corresponds to the stall force of the motor; the point at which, despite catalysis continuing, the untwisting force exerted by the entwined strands balances conformation selection in the motor’s catalytic cycle. Subsequent addition of the opposite enantiomeric fuelling system powers rotation of the motor-molecules of the contracted gel in the reverse direction, unwinding the entanglements and causing the gel to re-expand. Continued powered twisting of the strands in the new direction causes the gel to contract once again. The experimental demonstration of work against a load by a synthetic catalyst, and the mechanism of the transduction of energy by a catalyst through kinetic asymmetry in its acceleration of a fuel-to-waste reaction, informs both the debate surrounding the mechanism of force generation by biological motors and the design principles for artificial molecular nanotechnology.

Over the last two decades ratchet mechanisms have transformed the understanding and design of stochastic molecular systems—biological, chemical and physical—in a move away from the mechanical macroscopic analogies that dominated thinking regarding molecular dynamics in the 1990s and early 2000s (e.g. pistons, springs, etc), to the more scale‐relevant concepts that underpin out‐of‐equilibrium research in the molecular sciences today. Ratcheting has established molecular nanotechnology as a research frontier for energy transduction and metabolism, and has enabled the reverse engineering of biomolecular machinery, delivering insights into how molecules ‘walk’ and track‐based synthesisers operate, how the acceleration of chemical reactions enables energy to be transduced by catalysts (both motor proteins and synthetic catalysts), and how dynamic systems can be driven away from equilibrium through catalysis. The recognition of molecular ratchet mechanisms in biology, and their invention in synthetic systems, is proving significant in areas as diverse as supramolecular chemistry, systems chemistry, dynamic covalent chemistry, DNA nanotechnology, polymer and materials science, molecular biology, heterogeneous catalysis, endergonic synthesis, the origin of life, and many other branches of chemical science. Put simply, ratchet mechanisms give chemistry direction. Kinetic asymmetry, the key feature of ratcheting, is the dynamic counterpart of structural asymmetry (i.e. chirality). Given the ubiquity of ratchet mechanisms in endergonic chemical processes in biology, and their significance for behaviour and function from systems to synthesis, it is surely just as fundamentally important. This Review charts the recognition, invention and development of molecular ratchets, focussing particularly on the role for which they were originally envisaged in chemistry, as design elements for molecular machinery. Different kinetically asymmetric systems are compared, and the consequences of their dynamic behaviour discussed. These archetypal examples demonstrate how chemical systems can be driven inexorably away from equilibrium, rather than relax towards it.

Over the last two decades ratchet mechanisms have transformed the understanding and design of stochastic molecular systems—biological, chemical and physical—in a move away from the mechanical macroscopic analogies that dominated thinking regarding molecular dynamics in the 1990s and early 2000s (e.g. pistons, springs, etc), to the more scale‐relevant concepts that underpin out‐of‐equilibrium research in the molecular sciences today. Ratcheting has established molecular nanotechnology as a research frontier for energy transduction and metabolism, and has enabled the reverse engineering of biomolecular machinery, delivering insights into how molecules ‘walk’ and track‐based synthesisers operate, how the acceleration of chemical reactions enables energy to be transduced by catalysts (both motor proteins and synthetic catalysts), and how dynamic systems can be driven away from equilibrium through catalysis. The recognition of molecular ratchet mechanisms in biology, and their invention in synthetic systems, is proving significant in areas as diverse as supramolecular chemistry, systems chemistry, dynamic covalent chemistry, DNA nanotechnology, polymer and materials science, molecular biology, heterogeneous catalysis, endergonic synthesis, the origin of life, and many other branches of chemical science. Put simply, ratchet mechanisms give chemistry direction. Kinetic asymmetry, the key feature of ratcheting, is the dynamic counterpart of structural asymmetry (i.e. chirality). Given the ubiquity of ratchet mechanisms in endergonic chemical processes in biology, and their significance for behaviour and function from systems to synthesis, it is surely just as fundamentally important. This Review charts the recognition, invention and development of molecular ratchets, focussing particularly on the role for which they were originally envisaged in chemistry, as design elements for molecular machinery. Different kinetically asymmetric systems are compared, and the consequences of their dynamic behaviour discussed. These archetypal examples demonstrate how chemical systems can be driven inexorably away from equilibrium, rather than relax towards it.