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2.B.15 The Bis or Tris-thiourea Tripodal-based Chloride Carrier (TUT-CC) Family

The interaction of six tripodal synthetic chloride transmembrane transporters with a phosphatidyl choline (POPC) bilayer has been investigated by means of molecular dynamics simulations using the general Amber force field (GAFF) for the transporters and the LIPID11 force field for the phospholipids (Marques et al. 2014). These transporters are structurally simple molecules based on the tris(2-aminoethyl)amine scaffold, containing three thiourea binding units coupled with three n-butyl, phenyl, fluorophenyl, pentafluorophenyl, trifluoromethylphenyl, or bis(trifluoromethyl)phenyl substituents. The passive diffusion of Cl- was evaluated with the complexes initially positioned either in the water phase or inside the bilayer. In the first scenario, the chloride was released in the water solution before the synthetic molecules achieved the water-lipid interface and permeated the membrane. In the latter one, only when the chloride complex reached the interface was the anion released into the water phase, with the transporter losing the initial ggg tripodal shape. Independently of the transporter used in the membrane system, the bilayer structure was preserved, and the synthetic molecules interacted with the POPC molecules at the phosphate headgroup level via N-HO hydrogen bonds. Overall, the molecular dynamics simulations indicated that the small tripodal molecules in this series have a low impact on the bilayer and are able to diffuse with chloride inside the lipid environment. Indeed, these are essential conditions for these molecules to promote transmembrane transport as anion carriers, in agreement with experimental efflux data (Marques et al. 2014). 

Steroid-based cholapod anion carriers can bind anions (Cl- and NO3-) and carry them through the membrane. Activity is enhanced by placing electron withdrawing groups in the aromatic urea substituents, and by converting urea to thiourea (Valkenier and Davis 2013). Bis-thiourea derivatives have been prepared and tested for Cl-/NO3- exchange in 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol large unilamellar vesicles (LUVs). The bis-thioureas are typically >10 times more effective than the corresponding ureas. The highest activity was shown by decalin 9, which features N-(3,5-bis(trifluoromethyl)phenyl)thioureido and octyl ester substituents (Valkenier et al. 2014). A single molecule of transporter 9 in a 200 nm vesicle promoted Cl-/NO3- exchange with a half-life of 45 s and an absolute rate of 850 chloride anions per second. 

When compared with non-fluorinated analogues, fluorinated compounds demonstrate a different mechanism of membrane transport because the free transporter cannot diffuse through the membrane (Spooner et al. 2019). As a result, in H+/Cl- cotransport assays, fluorinated transporters require the presence of oleic acid to form anionic oleate complexes for recycling of the transporter, whereas non-fluorinated analogues readily diffuse through the membrane as free transporters and show synergistic transport with the proton transporter gramicidin. Molecular dynamics simulations revealed markedly stronger transporter-lipid interactions for fluorinated compounds compared with non-fluorinated analogues and hence, higher energy barriers for fluorinated compounds to cross the membrane as free transporters. With use of appropriate proton transporters to ensure measurement of the correct rate-limiting steps, the transport rates determined in synthetic vesicle assays show excellent agreement with the anion transport rates determined in cell-based assays (Spooner et al. 2019).

This family belongs to the: Thiourea Artificial Ion Transporter.

References associated with 2.B.15 family:

Marques I., Colaco AR., Costa PJ., Busschaert N., Gale PA. and Felix V. (2014). Tris-thiourea tripodal-based molecules as chloride transmembrane transporters: insights from molecular dynamics simulations. Soft Matter. 10(20):3608-21. 24663079
Spooner, M.J., H. Li, I. Marques, P.M.R. Costa, X. Wu, E.N.W. Howe, N. Busschaert, S.J. Moore, M.E. Light, D.N. Sheppard, V. FĂ©lix, and P.A. Gale. (2019). Fluorinated synthetic anion carriers: experimental and computational insights into transmembrane chloride transport. Chem Sci 10: 1976-1985. 30881627
Valkenier, H. and A.P. Davis. (2013). Making a match for Valinomycin: steroidal scaffolds in the design of electroneutral, electrogenic anion carriers. Acc Chem Res 46: 2898-2909. 23514113
Valkenier, H., L.W. Judd, H. Li, S. Hussain, D.N. Sheppard, and A.P. Davis. (2014). Preorganized bis-thioureas as powerful anion carriers: chloride transport by single molecules in large unilamellar vesicles. J. Am. Chem. Soc. 136: 12507-12512. 25122590