Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10⁻¹⁴ cm³/s or 3.5(±1.0) × 10⁸ water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10⁸ water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10⁸ water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10⁷ water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10⁵ pores per μm²) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10³ pores per μm²). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.The discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1–9). Areas of application include liquid and gas separations (10–13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen–Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The large-scale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water channels? (ii) How can such artificial channels be packaged into materials with morphologies suitable for engineering applications?Because of the challenges in accurately replicating the functional elements of channel proteins, the water permeability and selectivity of first-generation artificial water channels were far below those of AQPs (SI Appendix, Table S1) (20–25). In some cases, the conduction rate for water was much lower than that of AQPs as a result of excess hydrogen bonds being formed between the water molecules and oxygen atoms lining the channel (20). The low water permeability that was measured for first-generation water channels also highlights the experimental challenge of accurately characterizing water flow through low-permeability water channels. Traditionally, a liposome-based technique has been used to measure water conduction, in which the response to an osmotic gradient is followed by measuring changes in light scattering (26, 27) or fluorescence (28). The measured rates are then converted to permeability values. These measurements suffer from a high background signal due to water diffusion through the lipid bilayer, which, in some cases, can be higher than water conduction through the inserted channels, making it challenging to resolve the permeability contributed by the channels (29). Thus, there is a critical need for a method to accurately measure single-channel permeability of artificial water channels to allow for accurate comparison with those of biological water channels. Furthermore, first-generation artificial water channels were designed with a focus on demonstrating water conduction and one-dimensional assembly into tubular structures (20–24), but how the channels could be assembled into materials suitable for use in engineering applications was not explored. To derive the most advantage from their fast and selective transport properties, artificial water channels are ideally vertically aligned and densely packed in a flat membrane. These features have been long desired but remain a challenge for CNT-based systems (4).Here we introduce peptide-appended pillar5]arene (PAP; Fig. 1) (30) as an excellent architecture for artificial water channels, and we present data for their single-channel permeability and self-assembly properties. Nonpeptide pillar5]arene derivatives were among first-generation artificial water channels (1, 23). Pillar5]arene derivatives, including the one used in this study, have a pore of ∼5 Å in diameter and are excellent templates for functionalization into tubular structures (31–34). However, the permeability of hydrazide-appended pillar5]arene channels was low (∼6 orders of magnitude lower than that of AQPs; SI Appendix, Table S1). We addressed the challenges of accurately measuring single-channel water permeability and improving the water conduction rate over first-generation artificial water channels by using both experimental and simulation approaches. The presented PAP channel contains more hydrophobic regions (30) compared with its predecessor channel (23), which improves both its water permeability and its ability to insert into membranes. To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35, 36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36, 37). The combination of the two techniques allows molecular characterization of channel properties with high resolution and demonstrates that PAP channels have a water permeability close to those of AQPs and CNTs. The experimental results were corroborated by molecular dynamics (MD) simulations, which also provided additional insights into orientation and aggregation of the channels in lipid membranes. Finally, as a first step toward engineering applications such as liquid and gas separations, we were able to assemble PAP channels into highly packed planar membranes, and we experimentally confirmed that the channels form 2D arrays in these membranes.Open in a separate windowFig. 1.Structure of the peptide-appended pillar5]arene (PAP) channel. (A) The PAP channel (C₃₂₅H₃₂₀N₃₀O₆₀) forms a pentameric tubular structure through intramolecular hydrogen bonding between adjacent alternating d-l-d phenylalanine chains (d-Phe-l-Phe-d-Phe-COOH). (B) Molecular modeling (Gaussian09, semiempirical, PM6) of the PAP channel shows that the benzyl rings of the phenylalanine side chains extend outward from the channel walls (C, purple; H, white; O, red; N, blue). (C and D) MD simulation of the PAP channel in a POPC bilayer revealed its interactions with the surrounding lipids. The five chain-like units of the channel are colored purple, blue, ochre, green, and violet, with hydrogen atoms omitted. In C, the POPC lipids are represented by thin tan lines; in D, water is shown as red (oxygen) and white (hydrogen) van der Waals spheres.