LETTER doi:10.1038/nature10720 Pathway complexity in supramolecular polymerization Peter A. Korevaar 1,2 , Subi J. George 2 , Albert J. Markvoort 1,3 , Maarten M. J. Smulders 1,2 , Peter A. J. Hilbers 1,3 , Albert P. H. J. Schenning 2 , Tom F. A. De Greef 1,2,3 & E. W. Meijer 1,2 Self-assembly provides an attractive route to functional organic mate- rials, with properties and hence performance depending sensitively on the organization of the molecular building blocks 1–5 . Molecular organization is a direct consequence of the pathways involved in the supramolecular assembly process, which is more amenable to detailed study when using one-dimensional systems. In the case of protein fibrils, formation and growth have been attributed to complex aggregation pathways 6–8 that go beyond traditional concepts of homo- geneous 9–11 and secondary 12–14 nucleation events. The self-assembly of synthetic supramolecular polymers has also been studied and even modulated 15–18 , but our quantitative understanding of the processes involved remains limited. Here we report time-resolved observations of the formation of supramolecular polymers from p-conjugated oli- gomers. Our kinetic experiments show the presence of a kinetically favoured metastable assembly that forms quickly but then transforms into the thermodynamically favoured form. Quantitative insight into the kinetic experiments was obtained from kinetic model calculations, which revealed two parallel and competing pathways leading to assemblies with opposite helicity. These insights prompt us to use a chiral tartaric acid as an auxiliary to change the thermodynamic pref- erence of the assembly process 19 . We find that we can force aggrega- tion completely down the kinetically favoured pathway so that, on removal of the auxiliary, we obtain only metastable assemblies. The p-conjugated oligomer S-chiral oligo(p-phenylenevinylene) (SOPV, Fig 1a) serves as a functional material in a variety of organic electronic devices, with performance critically dependent on the material’s morphology 20 . This morphology is determined by the assembly mechanisms that transform the molecular dissolved monomers into the materials used in devices. The hydrogen-bonded dimers of SOPV self-assemble in apolar solvents via p–p interactions into one-dimensional helical stacks, and earlier circular dichroism (CD) and ultraviolet–visual spectroscopy studies under thermodynamic control revealed exclusive formation of left-handed M-type helical aggregates (M-SOPV) through a nucleated growth mechanism 21 . In practice, however, the assembly process is often under kinetic control—prompting us to examine in detail the supramolecular polymerization process under non-equilibrium conditions. Upon rapid quenching of SOPV from the molecularly dissolved state to 273K, we observed the formation of a mixture of M-SOPV and aggregates with opposite helicity, as evidenced by the opposite sign of the bisignated Cotton effect (Fig. 1b). At 298 K, these right-handed P-type aggregates (P-SOPV) slowly converted into thermodynamically stable M-SOPV aggregates (Supplementary Fig. 1). The observation of metastable P-SOPV aggregates indicated that the supramolecular polymerization of SOPV involves two different aggregation pathways, which we termed the on-pathway (leading to M-SOPV) and off- pathway (leading to P-SOPV) (Fig. 1c). To study the aggregation mechanism of SOPV and quantify the self- assembly pathways under kinetic control, we conducted stopped-flow experiments in which a concentrated solution of molecularly dissolved SOPV in chloroform was mixed with an excess of methylcyclohexane (MCH) to initiate self-assembly. The subsequent formation of helical SOPV aggregated in MCH was probed using CD spectroscopy (Supplementary Discussion 1). These kinetic experiments were con- ducted at 293 K and 308 K, with different concentrations of SOPV. At 293K and the lowest SOPV concentrations, the rate of aggregate formation initially increased with time, characteristic of a lag phase (Fig. 2a, c). The time-dependent CD signal was always negative under these conditions, suggesting the direct formation of thermodynamically stable on-pathway M-SOPV aggregates. At higher concentrations ($9 mM) a positive CD signal appeared in the initial stages of the assembly process and then developed into a negative CD signal at later times, suggesting the initial formation of off-pathway P-SOPV aggre- gates that then convert into thermodynamically stable M-SOPV aggre- gates. Remarkably, the time at which 50% of the aggregation process was completed (t 2 50) is longer at 15 mM than at 10 mM (Fig. 2d). Analogous kinetic studies at 308K also revealed the presence of a lag phase in experiments at the lowest concentrations (Fig. 2b, e) and an inverted dependence of t 2 50 on concentration, although the shortest t 2 50 time shifts to a higher SOPV concentration (20 mM; Fig. 2d). Detailed analyses unambiguously prove that both the on- and off- pathway are formed via a homogeneously nucleated growth mech- anism (Supplementary Figs 5 and 6). To rationalize the experimental aggregation kinetics, we extended models known in the field of protein fibrillization 6,9,11 by incorporating two competing, nucleated assembly pathways in which prenucleus oligomers (oligomer aggregates below the critical nucleation size) and helical aggregates change size through monomer association and dissociation (Fig. 3a, Supplementary Discussion 2). For the M-SOPV aggregates, non-helical prenucleus oligomers change size with rate constants for association and dissociation of a and b, respec- tively. Once the critical nucleus (with size n) is reached, the helical aggregates are in the elongation regime and change size through monomer association with the same rate constant a, while dissociation proceeds with rate constant c. The steady-state concentration of the aggregates in the nucleation phase is determined by the nucleation equilibrium constant K n 5 a/b, while in the elongation phase it is determined by the elongation equilibrium constant K e 5 a/c. The nucleated growth of off-pathway P-SOPV aggregates is described analogously, with nucleus size n*, rate constants a*, b* and c*, and equilibrium constants K n * and K e *. An essential model assumption is that the transition from metastable to thermodynamically stable aggre- gates occurs via depolymerization of P-SOPV aggregates and sub- sequent growth of M-SOPV aggregates. This is justified by the high helix reversal penalty (8.1 k B T, where k B is the Boltzmann constant and T is temperature), obtained by ‘majority rules’ experiments, which rules out intrastack stereomutation as an alternative transition mech- anism (Supplementary Fig. 8) 22 . 1 Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB, Eindhoven, The Netherlands. 2 Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, PO Box 513, 5600 MB, Eindhoven, The Netherlands. 3 Biomodeling and Bioinformatics Group, Eindhoven University of Technology, PO Box 513, 5600 MB, Eindhoven, The Netherlands. 492 | NATURE | VOL 481 | 26 JANUARY 2012 Macmillan Publishers Limited. All rights reserved ©2012