Nicolas Illy 1 , Laurent Bacri 2 , Loïc Auvray 2 , Jacques Penelle 1 and Valessa Barbier 1

1. Biosensing with channels 2007: channels, chips and nanopores Berder, 25-31 August 2007 Towards a novel polymeric ion channel Nicolas Illy1, Laurent Bacri2, Loïc Auvray2, Jacques Penelle1 and Valessa Barbier1 1Institut Chimie et Matériaux de Paris-Est (UMR 7182), CNRS-Thiais, équipe “Systèmes Polymères Complexes” 2Université d’Evry Val d’Essonne, Laboratoire Matériaux Polymère aux Interfaces

2. 2 Artificial ion channels based on crownether Mimic ion channel using crownether • Supramolecular Chemistry • lack of rigidity • Polypeptides • solid state synthesis : expensive, time-consuming • Polymer Chemistry • polydispersity A novel oligomeric structure using anionic living polymerization

3. 3 A novel oligomeric structure K+ K+ K+ ~5 Angström Anionic ring-opening polymerization of cyclopropane derivatives bearing crownether • Expected Structure: • rigid structure • crown-ether substituents: every third C-atom and cofacially stacked • low polydispersity (anionic polymerisation) • selective transport of metal cations 2 ion channels running parallel to main backbone

4. 4 Monomer synthesis c b a First step* : the crown ether obtained in a cyclisation reaction. Yield = 40 %. Second step : cyclopropanation with 1,2-dibromoethane. Yield = 60 %. Tfus = 67°C precursor a c b a monomer c b Vitreous solid Good purity DMSO *Bradshaw, JACS, 1976

5. 5 Ring opening polymerization • Synthesis : • Anionic polymerization • Inert atmosphere work (glovebox) • T° = 140°C • 3 days • Purification by precipitation in cold methanol Orange viscous liquid • Size exclusion chromatography in CHCl3 : Sample used for conductance experiment Mn = 6000 g.mol-1(estimation of the oligomer length = 50 Å) IP = 1.5 Futur work : polymerization livingness

6. 6 Planar bilayer conductance experiments Experimental device 3-5 nm V Oligomer sample KCl 1M 150 µm Cis Trans Membrane - Variable V • - Phospholipids: 1,2-diphytanoyl-glycero-3-phosphocholine (1%, decane) • Lipid membrane thickness: 4-8 nm • Oligomers solution in 1 M KCl solution • Two distinct behaviours: • Single isolated channel • Large aggregates

7. 7 Planar bilayer conductance experiments Ion channel behaviour • Measured intensity vs time (membrane thickness = 5.8 nm; Δ V = 100 mV) : I0 = 3.1 ± 0.33 pA I1 = 7.3 ± 0.56 pA I2 = 10 ± 1.0 pA I100mV = 3.58 pA I Single channel under 100 mV = 3.58 pA Electron flow: Q100mV = 2.24*107 ions.s-1 I50mV = 2.02 pA Transport performance similar to a natural single channel molecule

8. 8 Planar bilayer conductance experiments Ion channel behaviour • Estimation of the channel conductance Slope = G = 37 pS Linearly increasing of the unit current with the applied voltage The ion channel associated with the unit current follows Ohm’s Law • Estimation of the inner channel diameter With 0 = Vacuum permittivity = 8.854.10-12 F.m-1  = dielectric constant of a biological membrane = 2 S = nominal pinhole surface (150 μM) K = conductivity of 1 M KCl solution = 11.2 S.m-1 C = membrane capacitance D = 1.63*10-10 m = 1.63 Å

9. 9 Planar bilayer conductance experiments Aggregated channels • Great lifetime diversity Δ V = 100 mV Δ V = 100 mV Δ V = 100 mV < 1 ms ~ 1 to 5 ms > 200 s Unusually short-lifetime Long-lived aggregates Insertion difficulties • Amplitude diversity • The largest observed current reached 300 pA • Estimation of the equivalent diameter of the pores sizes : D = 14 Å

10. 10 Planar bilayer conductance experiments I0 = - 1.6 ± 1.7 pA I1 = - 55 ± 8.5 pA I2 = - 112 ± 9.7 pA Δ V = -100 mV The background noise increases with the number of aggregates • Coexistence between aggregates and single channels Δ V = 20 mV 40.6 ± 0.29 pA 42.2 ± 0.21 pA 2 behaviors simultaneously

11. 11 Conclusions • Synthesis of a new polycrownether • Insertion in lipid membranes Outlooks Cation selectivity studies Aggregation studies Size control of the polymer/ dependance on the length