Addressing Challenges in Ion-Selectivity Characterization in Nanopores.

Ion selectivity is the basis for designing smart nanopore/channel-based devices, e.g., ion separators and biosensors. Quantitative characterization of ion selectivities in nanopores often employs the Nernst or Goldman-Hodgkin-Katz (GHK) equation to interpret transmembrane potentials. However, the direction of the measured transmembrane potential drop is not specified in these equations, and selectivity values calculated using absolute values of transmembrane potentials do not directly reveal the ion for which the membrane is selective. Moreover, researchers arbitrarily choose whether to use the Nernst or GHK equation and overlook the significant differences between them, leading to ineffective quantitative comparisons between studies. This work addresses these challenges through (a) specifying the transmembrane potential (sign) and salt concentrations in terms of working and reference electrodes and the solutions in which they reside when using the Nernst and GHK equations, (b) reporting of both Nernst-selectivity and GHK-selectivity along with solution compositions and transmembrane potentials when comparing different nanopores/channels, and (c) performing simulations to define an ideal selectivity for nanochannels. Experimental and modeling studies provide significant insight into these fundamental equations and guidelines for the development of nanopore/channel-based devices.

Redox-Regulated Synthetic Channels: Enabling Reversible Ion Transport by Modulating the Ion-Permeation Pathway.

Natural redox-regulated channel proteins often utilize disulfide bonds as redox sensors for adaptive regulation of channel conformations in response to diverse physiological environments. In this study, we developed novel synthetic ion channels capable of reversibly switching their ion-transport capabilities by incorporating multiple disulfide bonds into artificial systems. X-ray structural analysis and electrophysiological experiments demonstrated that these disulfide-bridged molecules possess well-defined tubular cavities and can be efficiently inserted into lipid bilayers to form artificial ion channels. More importantly, the disulfide bonds in these molecules serve as redox-tunable switches to regulate the formation and disruption of ion-permeation pathways, thereby achieving a transition in the transmembrane transport process between the ON and OFF states.

Non-Covalently Stapled H+ /Cl- Ion Channels Activatable by Visible Light for Targeted Anticancer Therapy.

The development of stimuli-responsive artificial H+ /Cl- ion channels, capable of specifically disturbing the intracellular ion homeostasis of cancer cells, presents an intriguing opportunity for achieving high selectivity in cancer therapy. Herein, we describe a novel family of non-covalently stapled self-assembled artificial channels activatable by biocompatible visible light at 442 nm, which enables the co-transport of H+ /Cl- across the membrane with H+ /Cl- transport selectivity of 6.0. Upon photoirradiation of the caged C4F-L for 10 min, 90 % of ion transport efficiency can be restored, giving rise to a 10.5-fold enhancement in cytotoxicity against human colorectal cancer cells (IC50 =8.5 µM). The mechanism underlying cancer cell death mediated by the H+ /Cl- channels involves the activation of the caspase 9 apoptosis pathway as well as the scarcely reported disruption of the autophagic processes. In the absence of photoirradiation, C4F-L exhibits minimal toxicity towards normal intestine cells, even at a concentration of 200 µM.

Synthetic K+ Channels Constructed by Rebuilding the Core Modules of Natural K+ Channels in an Artificial System.

Different types of natural K+ channels share similar core modules and cation permeability characteristics. In this study, we have developed novel artificial K+ channels by rebuilding the core modules of natural K+ channels in artificial systems. All the channels displayed high selectivity for K+ over Na+ and exhibited a selectivity sequence of K+ ≈Rb+ during the transport process, which is highly consistent with the cation permeability characteristics of natural K+ channels. More importantly, these artificial channels could be efficiently inserted into cell membranes and mediate the transmembrane transport of K+ , disrupting the cellular K+ homeostasis and eventually triggering the apoptosis of cells. These findings demonstrate that, by rebuilding the core modules of natural K+ channels in artificial systems, the structures, transport behaviors, and physiological functions of natural K+ channels can be mimicked in synthetic channels.

Artificial K+ Channels Formed by Pillararene-Cyclodextrin Hybrid Molecules: Tuning Cation Selectivity and Generating Membrane Potential.

A class of artificial K+ channels formed by pillararene-cyclodextrin hybrid molecules have been designed and synthesized. These channels efficiently inserted into lipid bilayers and displayed high selectivity for K+ over Na+ in fluorescence and electrophysiological experiments. The cation transport selectivity of the artificial channels is tunable by varying the length of the linkers between pillararene and cyclodexrin. The shortest channel showed specific transmembrane transport preference for K+ over all alkali metal ions (selective sequence: K+ > Cs+ > Rb+ > Na+ > Li+ ), and is rarely observed for artificial K+ channels. The high selectivity of this artificial channel for K+ over Na+ ensures specific transmembrane translocation of K+ , and generated stable membrane potential across lipid bilayers.

ADP-ribosyl-N3: A Versatile Precursor for Divergent Syntheses of ADP-ribosylated Compounds.

Adenosine diphosphate-ribose (ADP-ribose) and its derivatives play important roles in a series of complex physiological procedures. The design and synthesis of artificial ADP-ribosylated compounds is an efficient way to develop valuable chemical biology tools and discover new drug candidates. However, the synthesis of ADP-ribosylated compounds is currently difficult due to structural complexity, easily broken pyrophosphate bond and high hydrophilicity. In this paper, ADP-ribosyl-N3 was designed and synthesized for the first time. With ADP-ribosyl-N3 as the key precursor, a divergent post-modification strategy was developed to prepare structurally diverse ADP-ribosylated compounds including novel nucleotides and peptides bearing ADP-ribosyl moieties.

Synthetic Channel Specifically Inserts into the Lipid Bilayer of Gram-Positive Bacteria but not that of Mammalian Erythrocytes.

A series of tubular molecules with different lengths have been synthesized by attaching Trp-incorporated peptides to the pillar[5]arene backbone. The tubular molecules are able to insert into the lipid bilayer to form unimolecular transmembrane channels. One of the channels has been revealed to specifically insert into the bilayer of the Gram-positive bacteria. In contrast, this channel cannot insert into the membranes of the mammalian rat erythrocytes even at the high concentration of 100 µm. It was further demonstrated that, as a result of this high membrane selectivity, the channel exhibits efficient antimicrobial activity for the Gram-positive bacteria and very low hemolytic toxicity for mammalian erythrocytes.

Tubular Unimolecular Transmembrane Channels: Construction Strategy and Transport Activities.

Lipid bilayer membranes separate living cells from their environment. Membrane proteins are responsible for the processing of ion and molecular inputs and exports, sensing stimuli and signals across the bilayers, which may operate in a channel or carrier mechanism. Inspired by these wide-ranging functions of membrane proteins, chemists have made great efforts in constructing synthetic mimics in order to understand the transport mechanisms, create materials for separation, and develop therapeutic agents. Since the report of an alkylated cyclodextrin for transporting Cu(2+) and Co(2+) by Tabushi and co-workers in 1982, chemists have constructed a variety of artificial transmembrane channels by making use of either the multimolecular self-assembly or unimolecular strategy. In the context of the design of unimolecular channels, important advances have been made, including, among others, the tethering of natural gramicidin A or alamethicin and the modification of various macrocycles such as crown ethers, cyclodextrins, calixarenes, and cucurbiturils. Many of these unimolecular channels exhibit high transport ability for metal ions, particularly K(+) and Na(+). Concerning the development of artificial channels based on macrocyclic frameworks, one straightforward and efficient approach is to introduce discrete chains to reinforce their capability to insert into bilayers. Currently, this approach has found the widest applications in the systems of crown ethers and calixarenes. We envisioned that for macrocycle-based unimolecular channels, control of the arrangement of the appended chains in the upward and/or downward direction would favor the insertion of the molecular systems into bilayers, while the introduction of additional interactions among the chains would further stabilize a tubular conformation. Both factors should be helpful for the formation of new efficient channels. In this Account, we discuss our efforts in designing new unimolecular artificial channels from tubular pillar[n]arenes by extending their lengths with various ester, hydrazide, and short peptide chains. We have utilized well-defined pillar[5]arene and pillar[6]arene as rigid frameworks that allow the appended chains to afford extended tubular structures. We demonstrate that the hydrazide and peptide chains form intramolecular N-H···O═C hydrogen bonds that enhance the tubular conformation of the whole molecule. The new pillar[n]arene derivatives have been successfully applied as unimolecular channels for the selective transport of protons, water, and amino acids and the voltage-gated transport of K(+). We also show that aromatic hydrazide helices and macrocycles appended with peptide chains are able to mediate the selective transport of NH4(+).

Hydrogen-bonded helical hydrazide oligomers and polymer that mimic the ion transport of gramicidin A.

A new series of hydrogen-bonded helical aromatic hydrazide oligomers and polymer that bear phenylalanine tripeptide chains have been designed and synthesized. It was revealed that the helical structures could insert into lipid bilayers to form unimolecular channels. The longest oligomeric and polymeric helical channels exhibited an NH4(+)/K(+) selectivity that was higher than that of natural gramicidin A, whereas the transport of a short helical channel for Tl(+) could achieve an efficiency as high as that of gramicidin A.

Effect of charge status on the ion transport and antimicrobial activity of synthetic channels.

A class of unimolecular channels formed by pillararene-gramicidin hybrid molecules are presented. The charge status of the peptide domain in these channels has a significant impact on their ion transport and antimicrobial activity. These channels exhibited different membrane-association abilities between microbial cells and mammalian cells. One of the channels displayed a higher antimicrobial activity towards S. aureus (IC50 = 0.55 µM) and negligible hemolytic toxicity, showing potential to serve as a systemic antibiotic.

A dual-responsive hyperbranched supramolecular polymer constructed by cooperative host-guest recognition and hydrogen-bond interactions.

A homotritopic pillar[5]arene (H3) containing adenine units was synthesized and employed to interact with a uracil derivative (6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)hexanenitrile, G) to form a hyperbranched supramolecular polymer. The hyperbranched supramolecular polymer showed a dual stimulus response both to heat and acid/base. The cooperative host-guest binding and hydrogen-bond interactions play a key role in the supramolecular polymerization.

One-pot formation of hydrazide macrocycles with modified cavities: an example of pH-sensitive unimolecular cation channels.

A series of pH-sensitive, cation-selective hydrazide macrocyclic channels have been synthesized. The macrocyclic channels bear multiple carboxyls in the inner cavity, which have a significant impact on their membrane-incorporation ability and NH4+ transport activity. Moreover, the K+/Cl- selectivities of the macrocyclic channels can be tuned by the pH value of the electrolyte.

Functionalized hydrazide macrocycle ion channels showing pH-sensitive ion selectivities.

Transmembrane channels formed by functionalized hydrazide macrocycles are reported. The different pH values of buffer solutions have a significant effect on the K+/Cl- selectivity of the macrocycles. This unique transport behavior is mainly induced by the different distributions of charges in the tubular channels under various pH values.

A unimolecular channel formed by dual helical peptide modified pillar[5]arene: correlating transmembrane transport properties with antimicrobial activity and haemolytic toxicity.

Five unimolecular channels with different lengths are presented. The varying length of these channels has significant impact on their transmembrane transport properties, which are directly correlated with their antimicrobial activity and inversely correlated with their haemolytic toxicity. By further structural optimization, these new channels could reach high antimicrobial activity and very low haemolytic toxicity, with the potential to serve as systemic antibiotics.

Hydrazide macrocycles as effective transmembrane channels for ammonium.

Three shape-persistent aromatic hydrazide macrocycles that bear phenylalanine tripeptide chains have been synthesized. These macrocycles can insert into lipid bilayers to form single-molecular ion channels which exhibit a high NH4(+)/K(+) selectivity.

A four-unit [c2]daisy chain connected by hydrogen bonds.

A mono-adenine-functionalized pillar[5]arene and a guest including uracil were prepared. They formed a novel four-unit [c2]daisy chain both in the solid state and in a chloroform solution. As far as we know, this [c2]daisy chain is the first one without a covalently bound linear thread. This unique assembly behavior is mainly induced by hydrogen-bond interactions between A and U in the A-U base pairs.

Nickel catalyzed alkylation of N-aromatic heterocycles with Grignard reagents through direct C-H bond functionalization.

A novel protocol for nickel-catalyzed direct sp(2) C-H bond alkylation of N-aromatic heterocycles has been developed. This new reaction proceeded efficiently at room temperature using a Grignard reagent as the coupling partner. This approach provides new access to a variety of alkylated N-aromatic heterocycles which are potentially of great importance in medicinal chemistry.

Nickel-catalyzed sp2 C-H bonds arylation of N-aromatic heterocycles with Grignard reagents at room temperature.

A novel protocol for nickel-catalyzed direct sp(2) C-H bond arylation of purines has been developed. This new reaction proceeded efficiently at room temperature using Grignard reagent as the coupling partner within 5 hours in good to high yields. This approach provides a new access to a variety of C8-arylpurines which are potentially of great importance in medicinal chemistry.