Periodic Reporting for period 1 - MOTOTRANS (Light-driven molecular motors to trigger transmembrane transport of anions)
Reporting period: 2024-07-01 to 2026-06-30
Summary of the context and overall objectives of the project
The transport of ions across cell membranes is essential for many biological processes and is normally controlled by highly sophisticated membrane proteins. Dysfunction of these natural transport systems is associated with a range of diseases. Synthetic molecules capable of transporting ions across lipid membranes therefore attract considerable interest, both as simplified models of biological transport systems and because of their potential future applications in areas such as therapeutics, sensing and functional membrane materials.
A major limitation of many synthetic ion transporters is that their activity cannot be readily controlled after incorporation into a membrane. Light offers an attractive external stimulus because it can provide precise spatial and temporal control without requiring the addition of chemical reagents. MOTOTRANS aimed to address this challenge by integrating artificial anion transporters with light-driven molecular motors. These molecular machines undergo controlled structural changes and directional rotary motion upon irradiation and therefore offered the possibility of using molecular-scale dynamics to regulate transport across lipid bilayer membranes.
The overall objective of MOTOTRANS was to develop light-responsive systems for transmembrane anion transport based on molecular motors and to understand how light-induced changes in these motors influence transport activity. Three complementary approaches were originally envisaged: combining molecular motors with separate anion carriers within lipid membranes; covalently linking an anion-binding carrier to a molecular motor; and developing motor-functionalised building blocks capable of forming transmembrane channels.
The project successfully demonstrated that a molecular motor equipped with an anion-binding thiourea unit can transport chloride ions across lipid bilayers and that its transport activity can be enhanced by light irradiation. Importantly, mechanistic investigations revealed that this enhancement is not caused by the rotary motion of the molecular motor itself, as initially hypothesised. Instead, the results indicate that photoisomerisation affects the aggregation and mobility of the transporter and modifies properties of the surrounding lipid membrane. This finding provides an important mechanistic basis for the future design of light-responsive artificial membrane transport systems.
MOTOTRANS therefore contributes to the broader development of controllable molecular machines and responsive supramolecular systems. In the longer term, the ability to regulate synthetic membrane transport with external stimuli could contribute to the development of advanced functional materials and artificial membrane systems, and may provide concepts relevant to sensing, delivery and biomedical applications. At the present stage, however, the project represents fundamental research, and its principal impact is the generation of new knowledge, methodologies and design principles for molecular-machine-based artificial ion transport.
No social sciences and humanities disciplines were required for the research objectives of MOTOTRANS.
A major limitation of many synthetic ion transporters is that their activity cannot be readily controlled after incorporation into a membrane. Light offers an attractive external stimulus because it can provide precise spatial and temporal control without requiring the addition of chemical reagents. MOTOTRANS aimed to address this challenge by integrating artificial anion transporters with light-driven molecular motors. These molecular machines undergo controlled structural changes and directional rotary motion upon irradiation and therefore offered the possibility of using molecular-scale dynamics to regulate transport across lipid bilayer membranes.
The overall objective of MOTOTRANS was to develop light-responsive systems for transmembrane anion transport based on molecular motors and to understand how light-induced changes in these motors influence transport activity. Three complementary approaches were originally envisaged: combining molecular motors with separate anion carriers within lipid membranes; covalently linking an anion-binding carrier to a molecular motor; and developing motor-functionalised building blocks capable of forming transmembrane channels.
The project successfully demonstrated that a molecular motor equipped with an anion-binding thiourea unit can transport chloride ions across lipid bilayers and that its transport activity can be enhanced by light irradiation. Importantly, mechanistic investigations revealed that this enhancement is not caused by the rotary motion of the molecular motor itself, as initially hypothesised. Instead, the results indicate that photoisomerisation affects the aggregation and mobility of the transporter and modifies properties of the surrounding lipid membrane. This finding provides an important mechanistic basis for the future design of light-responsive artificial membrane transport systems.
MOTOTRANS therefore contributes to the broader development of controllable molecular machines and responsive supramolecular systems. In the longer term, the ability to regulate synthetic membrane transport with external stimuli could contribute to the development of advanced functional materials and artificial membrane systems, and may provide concepts relevant to sensing, delivery and biomedical applications. At the present stage, however, the project represents fundamental research, and its principal impact is the generation of new knowledge, methodologies and design principles for molecular-machine-based artificial ion transport.
No social sciences and humanities disciplines were required for the research objectives of MOTOTRANS.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
The experimental work in MOTOTRANS focused on the development and investigation of molecular-motor-based systems for light-responsive transmembrane anion transport. The project involved the synthesis and characterization of Feringa-type molecular motors and anion transporters, investigation of their photochemical and thermal isomerization behaviour, and evaluation of their transport properties in model lipid bilayer membranes.
Initial work explored whether molecular motors incorporated into lipid membranes could enhance the activity of separate anion carriers upon light irradiation (WP1). Although the required molecular-motor and membrane-transport methodologies were established, the anticipated enhancement of transport by a separately doped motor/carrier system was not demonstrated. The project therefore focused on the complementary strategy of covalently linking an anion-binding unit to the molecular motor (WP2).
A fluorene-based molecular motor functionalized with a phenylthiourea anion-binding motif was successfully synthesized and characterized. Its photoisomerization and thermal relaxation were investigated by UV–Vis and NMR spectroscopy, and chloride binding was quantified by 1H NMR titration. The stable transporter showed a 1:1 chloride association constant of 1.9 × 10^3 M^−1 in CD3CN. Its transmembrane chloride transport activity was studied in POPC lipid vesicles using complementary chloride-selective-electrode and osmotic-response/light-scattering assays, the latter enabling transport to be monitored during light irradiation.
A major achievement was the demonstration of light-enhanced chloride transport. Irradiation at 405 nm generated a metastable state of the molecular motor that was more active in transporting chloride than the stable state. Dose-response experiments gave EC50 values of 4.62 mol% for the stable transporter and 2.59 mol% for the irradiated photostationary-state mixture, corresponding to at least a 1.8-fold increase in transport efficiency. Under in situ 405 nm irradiation, chloride transport was enhanced by up to 85% compared with the corresponding dark experiment. Control experiments confirmed that the effect required the thiourea anion-binding site and was not caused by nonspecific membrane leakage.
An important outcome of the project was clarification of the mechanism underlying this light-induced enhancement. A structurally related thioxanthene-based molecular motor with a much faster rotational rate was synthesized and investigated as a mechanistic control. Despite its rapid rotary motion, this analogue showed no discernible light-induced enhancement of chloride transport. The results therefore demonstrate that the enhancement observed for the slower motor is not caused by molecular-motor rotation itself. Further experiments indicated that photoisomerization affects aggregation and transporter mobility within the membrane and can alter the surrounding membrane environment. Consistent with this interpretation, incorporation of cholesterol into the lipid bilayer attenuated the light-induced enhancement.
This mechanistic finding represented an important refinement of the original project hypothesis. Consequently, the remaining project effort was prioritised toward rigorous characterization and mechanistic validation of the successful WP2 system rather than initiating the originally proposed motor-functionalized self-assembled channel studies (WP3). Overall, MOTOTRANS successfully established a molecular-motor-based artificial anion carrier whose transmembrane chloride transport can be enhanced by light and, importantly, demonstrated that this enhancement originates from photoisomer-dependent changes in transporter mobility/aggregation and membrane properties rather than from the rotary motion of the molecular motor itself.
Initial work explored whether molecular motors incorporated into lipid membranes could enhance the activity of separate anion carriers upon light irradiation (WP1). Although the required molecular-motor and membrane-transport methodologies were established, the anticipated enhancement of transport by a separately doped motor/carrier system was not demonstrated. The project therefore focused on the complementary strategy of covalently linking an anion-binding unit to the molecular motor (WP2).
A fluorene-based molecular motor functionalized with a phenylthiourea anion-binding motif was successfully synthesized and characterized. Its photoisomerization and thermal relaxation were investigated by UV–Vis and NMR spectroscopy, and chloride binding was quantified by 1H NMR titration. The stable transporter showed a 1:1 chloride association constant of 1.9 × 10^3 M^−1 in CD3CN. Its transmembrane chloride transport activity was studied in POPC lipid vesicles using complementary chloride-selective-electrode and osmotic-response/light-scattering assays, the latter enabling transport to be monitored during light irradiation.
A major achievement was the demonstration of light-enhanced chloride transport. Irradiation at 405 nm generated a metastable state of the molecular motor that was more active in transporting chloride than the stable state. Dose-response experiments gave EC50 values of 4.62 mol% for the stable transporter and 2.59 mol% for the irradiated photostationary-state mixture, corresponding to at least a 1.8-fold increase in transport efficiency. Under in situ 405 nm irradiation, chloride transport was enhanced by up to 85% compared with the corresponding dark experiment. Control experiments confirmed that the effect required the thiourea anion-binding site and was not caused by nonspecific membrane leakage.
An important outcome of the project was clarification of the mechanism underlying this light-induced enhancement. A structurally related thioxanthene-based molecular motor with a much faster rotational rate was synthesized and investigated as a mechanistic control. Despite its rapid rotary motion, this analogue showed no discernible light-induced enhancement of chloride transport. The results therefore demonstrate that the enhancement observed for the slower motor is not caused by molecular-motor rotation itself. Further experiments indicated that photoisomerization affects aggregation and transporter mobility within the membrane and can alter the surrounding membrane environment. Consistent with this interpretation, incorporation of cholesterol into the lipid bilayer attenuated the light-induced enhancement.
This mechanistic finding represented an important refinement of the original project hypothesis. Consequently, the remaining project effort was prioritised toward rigorous characterization and mechanistic validation of the successful WP2 system rather than initiating the originally proposed motor-functionalized self-assembled channel studies (WP3). Overall, MOTOTRANS successfully established a molecular-motor-based artificial anion carrier whose transmembrane chloride transport can be enhanced by light and, importantly, demonstrated that this enhancement originates from photoisomer-dependent changes in transporter mobility/aggregation and membrane properties rather than from the rotary motion of the molecular motor itself.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Prior to MOTOTRANS, light-driven molecular motors had been incorporated into synthetic membrane transport systems, particularly for alkali-metal cation transport, and enhanced transport under continuous irradiation had been associated with molecular-motor activity. However, the origin of this enhancement and, specifically, the role of directional rotary motion remained insufficiently understood. Furthermore, analogous molecular-motor-based systems for controlling transmembrane anion transport had not been established.
MOTOTRANS advances the state of the art by demonstrating light-enhanced transmembrane chloride transport using a Feringa-type molecular motor covalently functionalized with a thiourea anion-binding unit. Irradiation with 405 nm light generated a more active metastable state and enhanced chloride transport by up to 85%. This extends the application of light-driven molecular motors from previously investigated cation-transport systems to the control of anion transport across lipid bilayers.
More importantly, the project provides new mechanistic insight into how molecular motors can regulate membrane transport. A structurally related motor-based transporter capable of much faster rotary motion did not show light-enhanced transport. This demonstrates that, in the MOTOTRANS system, the enhancement cannot be attributed directly to the rotational motion or rotational speed of the molecular motor. Instead, the combined experimental evidence indicates that photoisomerization modifies aggregation and mobility of the transporter within the lipid bilayer and influences the surrounding membrane properties. The reduced light-induced enhancement observed upon incorporation of cholesterol into the membrane further supports the involvement of membrane organization and fluidity.
These findings provide an important design principle for future molecular-machine-based membrane transport systems: light-responsive transport does not necessarily require rapid molecular rotation, and control over photoinduced molecular state, aggregation, mobility and membrane organization can instead be exploited to regulate transport. The project therefore contributes not only a new light-responsive anion transporter but also a mechanistic framework for the design and interpretation of future artificial membrane transport systems.
The results are currently at the level of fundamental research, and no immediate commercial exploitation or intellectual-property protection is envisaged. Further uptake will primarily require additional fundamental research to establish how generally the identified mechanism applies to other molecular motors, transporter structures, lipid compositions and biologically relevant membrane environments. Future work could use these principles to develop systems with larger and more predictable light responses and improved control over membrane localization and transport activity. In the longer term, such externally controlled membrane transport systems could provide concepts relevant to responsive materials, artificial membranes, sensing and delivery technologies. At the present stage, however, the principal impact of MOTOTRANS is the generation of new knowledge, experimental methodologies and molecular-design principles that can guide further research in molecular machines and artificial ion transport.
This is also consistent with your approved exploitation plan, which classifies the results at TRL 1–2 and identifies the main exploitable outcomes as knowledge, methodologies and design principles rather than a commercial product.
MOTOTRANS advances the state of the art by demonstrating light-enhanced transmembrane chloride transport using a Feringa-type molecular motor covalently functionalized with a thiourea anion-binding unit. Irradiation with 405 nm light generated a more active metastable state and enhanced chloride transport by up to 85%. This extends the application of light-driven molecular motors from previously investigated cation-transport systems to the control of anion transport across lipid bilayers.
More importantly, the project provides new mechanistic insight into how molecular motors can regulate membrane transport. A structurally related motor-based transporter capable of much faster rotary motion did not show light-enhanced transport. This demonstrates that, in the MOTOTRANS system, the enhancement cannot be attributed directly to the rotational motion or rotational speed of the molecular motor. Instead, the combined experimental evidence indicates that photoisomerization modifies aggregation and mobility of the transporter within the lipid bilayer and influences the surrounding membrane properties. The reduced light-induced enhancement observed upon incorporation of cholesterol into the membrane further supports the involvement of membrane organization and fluidity.
These findings provide an important design principle for future molecular-machine-based membrane transport systems: light-responsive transport does not necessarily require rapid molecular rotation, and control over photoinduced molecular state, aggregation, mobility and membrane organization can instead be exploited to regulate transport. The project therefore contributes not only a new light-responsive anion transporter but also a mechanistic framework for the design and interpretation of future artificial membrane transport systems.
The results are currently at the level of fundamental research, and no immediate commercial exploitation or intellectual-property protection is envisaged. Further uptake will primarily require additional fundamental research to establish how generally the identified mechanism applies to other molecular motors, transporter structures, lipid compositions and biologically relevant membrane environments. Future work could use these principles to develop systems with larger and more predictable light responses and improved control over membrane localization and transport activity. In the longer term, such externally controlled membrane transport systems could provide concepts relevant to responsive materials, artificial membranes, sensing and delivery technologies. At the present stage, however, the principal impact of MOTOTRANS is the generation of new knowledge, experimental methodologies and molecular-design principles that can guide further research in molecular machines and artificial ion transport.
This is also consistent with your approved exploitation plan, which classifies the results at TRL 1–2 and identifies the main exploitable outcomes as knowledge, methodologies and design principles rather than a commercial product.