Functionalised nanopores: chemical and biological modifications

Nanopore technology has established itself as a powerful tool for single-molecule studies. By analysing changes in the ion current flowing through a single transmembrane channel, a wealth of molecular information can be elucidated. Early studies utilised nanopore technology for sensing applications, and subsequent developments have diversified its remit. Nanopores can be synthetic, solid-state, or biological in origin, but recent work has seen these boundaries blurred as hybrid functionalised pores emerge. The modification of existing pores and the construction of novel synthetic pores has been an enticing goal for creating systems with tailored properties and functionality. Here, we explore chemically functionalised biological pores and the bio-inspired functionalisation of solid-state pores, highlighting how the convergence of these domains provides enhanced functionality.

The convergence of chemistry, biology, and solid-state approaches enables the construction hybrid nanopores with enhanced single-molecule applications.     

Synthetic glycosidases for the precise hydrolysis of oligosaccharides and polysaccharides

Glycosidases are an important class of enzymes for performing the selective hydrolysis of glycans. Although glycans can be hydrolyzed in principle by acidic water, hydrolysis with high selectivity using nonenzymatic catalysts is an unachieved goal. Molecular imprinting in cross-linked micelles afforded water-soluble polymeric nanoparticles with a sugar-binding boroxole in the imprinted site. Post-modification installed an acidic group near the oxygen of the targeted glycosidic bond, with the acidity and distance of the acid varied systematically. The resulting synthetic glycosidase hydrolyzed oligosaccharides and polysaccharides in a highly controlled fashion simply in hot water. These catalysts not only broke down amylose with similar selectivities to those of natural enzymes, but they also could be designed to possess selectivity not available with biocatalysts. Substrate selectivity was mainly determined by the sugar residues bound within the active site, including their spatial orientations. Separation of the product was accomplished through in situ dialysis, and the catalysts left behind could be used multiple times with no signs of degradation. This work illustrates a general method to construct synthetic glycosidases from readily available building blocks via self-assembly, covalent capture, and post-modification. In addition, controlled, precise, one-step hydrolysis is an attractive way to prepare complex glycans from naturally available carbohydrate sources.

Synthetic glycosidases with a sugar-binding active site and a precisely positioned acidic group hydrolyze oligo- and polysaccharides selectively in hot water to afford desired sugar products in a single step.     

Fluorescent small organic probes for biosensing

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems. With this perspective we highlight sensing mechanisms including Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited state intramolecular proton transfer (ESIPT), aggregation induced emission (AIE) and multiple modality fluorescence approaches including dual/triple sensing mechanisms (DSM or TSM). Throughout the perspective we highlight the remaining challenges and suggest potential directions for development towards improved small-molecule fluorescent probes suitable for biosensing.

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems.     

Single-molecule nanopore sensing of actin dynamics and drug binding

Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed the unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopore sensing for in-depth understanding of protein folding landscapes and for drug discovery.

Nanopipettes were used for real-time investigation into actin dynamics and drug binding at single-molecule resolution, showing promise for a better understanding of the mechanism of protein–protein interactions and drug discovery.     

Metallohelices emulate the properties of short cationic α-helical peptides

Naturally occurring peptides in many living systems perform antimicrobial and anticancer host defence roles, but their potential for clinical application is limited by low metabolic stability and relatively high costs of goods. Self-assembled helical metal complexes provide an attractive synthetic platform for non-peptidic architectures that can emulate some of the properties of short cationic α-helical peptides, with tuneable charge, shape, size and amphipathicity. Correspondingly there is a growing body of evidence demonstrating that these supramolecular architectures exhibit bioactivity that emulates that of the natural systems. We review that evidence in the context of synthetic advances in the area, driven by the potential for biomedical applications. We note some design considerations for new biologically-relevant metallohelices, and give our outlook on the future of these compounds as therapeutic peptidomimetics.

Advances in metal–organic self-assembly have unearthed an area of chemical space containing peptide-emulating molecules. Comparison with natural systems indicates future directions in terms of synthesis, disease areas and mechanistic understanding.     

Directional asymmetry over multiple length scales in reticular porous materials

In nature and synthetic materials, asymmetry is a useful tool to create complex and functional systems constructed from a limited number of building blocks. Reticular chemistry has allowed the synthesis of a wide range of discrete and extended structures, from which modularity permits the controlled assembly of their constituents to generate asymmetric configurations of pores or architectures. In this perspective, we present the different strategies to impart directional asymmetry over nano/meso/macroscopic length scales in porous materials and the resulting novel properties and applications.

Design strategies for the controlled assembly of discrete and extended reticular materials with asymmetric configurations of pores or architectures.     

Debugging: putting the synthetic yeast chromosome to work

Synthetic genomics aims to de novo synthesize a functional genome redesigned from natural sequences with custom features. Designed genomes provide new toolkits for better understanding organisms, evolution and the construction of cellular factories. Currently maintaining the fitness of cells with synthetic genomes is particularly challenging as defective designs and unanticipated assembly errors frequently occur. Mapping and correcting bugs that arise during the synthetic process are imperative for the successful construction of a synthetic genome that can sustain a desired cellular function. Here, we review recently developed methods used to map and fix various bugs which arise during yeast genome synthesis with the hope of providing guidance for putting the synthetic yeast chromosome to work.

This review summarizes strategies used to map and repair various bugs in synthetic genomic sequences and provides guidance for the construction of synthetic yeast chromosomes that are capable of maintaining cell fitness.     

Efficient cleavage of tertiary amide bonds via radical–polar crossover using a copper(ii) bromide/Selectfluor hybrid system

A novel approach for the efficient cleavage of the amide bonds in tertiary amides is reported. Based on the selective radical abstraction of a benzylic hydrogen atom by a CuBr₂/Selectfluor hybrid system followed by a selective cleavage of an N–C bond, an acyl fluoride intermediate is formed. This intermediate may then be derivatized in a one-pot fashion. The reaction proceeds under mild conditions and exhibits a broad substrate scope with respect to the tertiary amide moiety as well as to nitrogen, oxygen, and carbon nucleophiles for the subsequent derivatization. Mechanistic studies suggest that the present reaction proceeds via a radical–polar crossover process that involves benzylic carbon radicals generated by the selective radical abstraction of a benzylic hydrogen atom by the CuBr₂/Selectfluor hybrid system. Furthermore, a synthetic application of this method for the selective cleavage of peptides is described.

A novel approach for the efficient cleavage of the amide bonds in tertiary amides is reported.     

Solid-phase fluorescent BODIPY–peptide synthesis via in situ dipyrrin construction

Traditional fluorescent peptide chemical syntheses hinge on the use of limited fluorescent/dye-taggable unnatural amino acids and entail multiple costly purifications. Here we describe a facile and efficient protocol for in situ construction of dipyrrins on the N-terminus with 20 natural and five unnatural amino acids and the lysine''s side chain of selected peptides/peptide drugs through Fmoc-based solid-phase peptide synthesis. The new strategy enables the direct formation of boron–dipyrromethene (BODIPY)–peptide conjugates from simple aldehyde and pyrrole derivatives without pre-functionalization, and only requires a single-time chromatographic purification at the final stage. As a model study, synthesized EBNA1-targeting BODIPY1–Pep4 demonstrates intact selectivity in vitro, responsive fluorescence enhancement, and higher light cytotoxicity due to the photo-generation of cytotoxic singlet oxygen. This work offers a novel practical synthetic platform for fluorescent peptides for multifaceted biomedical applications.

Solid-phase fluorescent BODIPY–peptide synthesis via in situ dipyrrin construction offers an efficient fluorescent peptide synthetic platform for multifaceted biomedical applications.     

Transmembrane anion transport mediated by halogen bonding and hydrogen bonding triazole anionophores

Transmembrane ion transport by synthetic anionophores is typically achieved using polar hydrogen bonding anion receptors. Here we show that readily accessible halogen and hydrogen bonding 1,2,3-triazole derivatives can efficiently mediate anion transport across lipid bilayer membranes with unusual anti-Hofmeister selectivity. Importantly, the results demonstrate that the iodo-triazole systems exhibit the highest reported activity to date for halogen bonding anionophores, and enhanced transport efficiency relative to the hydrogen bonding analogues. In contrast, the analogous fluoro-triazole systems, which are unable to form intermolecular interactions with anions, are inactive. The halogen bonding anionophores also exhibit a remarkable intrinsic chloride over hydroxide selectivity, which is usually observed only in more complex anionophore designs, in contrast to the readily accessible acyclic systems reported here. This highlights the potential of iodo-triazoles as synthetically accessible and versatile motifs for developing more efficient anion transport systems. Computational studies provide further insight into the nature of the anion-triazole intermolecular interactions, examining the origins of the observed transport activity and selectivity of the systems, and revealing the role of enhanced charge delocalisation in the halogen bonding anion complexes.

Halogen and hydrogen bonding 1,2,3-triazole derivatives efficiently mediate anion transport across lipid bilayer membranes with unusual anion selectivity profiles.     

Advances in anion binding and sensing using luminescent lanthanide complexes

Luminescent lanthanide complexes have been actively studied as selective anion receptors for the past two decades. Ln(iii) complexes, particularly of europium(iii) and terbium(iii), offer unique photophysical properties that are very valuable for anion sensing in biological media, including long luminescence lifetimes (milliseconds) that enable time-gating methods to eliminate background autofluorescence from biomolecules, and line-like emission spectra that allow ratiometric measurements. By careful design of the organic ligand, stable Ln(iii) complexes can be devised for rapid and reversible anion binding, providing a luminescence response that is fast and sensitive, offering the high spatial resolution required for biological imaging applications. This review focuses on recent progress in the development of Ln(iii) receptors that exhibit sufficiently high anion selectivity to be utilised in biological or environmental sensing applications. We evaluate the mechanisms of anion binding and sensing, and the strategies employed to tune anion affinity and selectivity, through variations in the structure and geometry of the ligand. We highlight examples of luminescent Ln(iii) receptors that have been utilised to detect and quantify specific anions in biological media (e.g. human serum), monitor enzyme reactions in real-time, and visualise target anions with high sensitivity in living cells.

This minireview highlights advances in anion binding and sensing using luminescent lanthanide(iii) complexes.     

Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores

Protein–protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.

Using aerolysin nanopores, we probed protein–protein interaction (PPI) between p53TAD and MDM2 and its modulation by small-molecule PPI inhibitors and p53TAD phosphorylation.     

Construction of diverse peptide structural architectures via chemoselective peptide ligation

Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural architectures via chemoselective peptide ligation. The key advancement involved is to utilize the benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as the hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural design of PPI inhibitors with new modalities.

Methods of introducing peptide salicylaldehyde esters and hydroxyl amine functionality into the peptide side chain have been developed. Diverse peptide structural motifs were constructed via ligation with native amide linkages at the ligation sites.     

Synthetic half-reactions

This perspective on reactivity introduces Synthetic Half-Reactions (SHRs) as a way to analyze chemical transformations. SHRs denote either an uphill transformation leading to a higher energy state or a downhill transformation leading to a lower energy state. Using well-established processes, I show how the matching of different classes of SHRs offers a tool to classify chemical transformations. This raises the possibility to discover new processes by finding underappreciated combinations of endergonic and exergonic steps.

This perspective on reactivity introduces Synthetic Half-Reactions (SHRs) as a way to analyze chemical transformations.     

A reconstructed porous copper surface promotes selectivity and efficiency toward C2 products by electrocatalytic CO2 reduction

Electrocatalytic synthesis of multicarbon (C₂₊) products from CO₂ reduction suffers from poor selectivity and low energy efficiency. Herein, a facile oxidation–reduction cycling method is adopted to reconstruct the Cu electrode surface with the help of halide anions. The surface composed of entangled Cu nanowires with hierarchical pores is synthesized in the presence of I⁻, exhibiting a C₂ faradaic efficiency (FE) of 80% at −1.09 V vs. RHE. A partial current density of 21 mA cm⁻² is achieved with a C₂ half-cell power conversion efficiency (PCE) of 39% on this electrode. Such high selective C₂ production is found to mainly originate from CO intermediate enrichment inside hierarchical pores rather than the surface lattice effect of the Cu electrode.

The Cu electrode surface is reconstructed by a halide anion assisted method for promoting CO₂ reduction.     

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport

Controlled design of giant unilamellar vesicles under defined conditions has vast applications in the field of membrane and synthetic biology. Here, we bio-engineer bacterial-membrane mimicking models of controlled size under defined salt conditions over a range of pH. A complex bacterial lipid extract is used for construction of physiologically relevant Gram-negative membrane mimicking vesicles whereas a ternary mixture of charged lipids (DOPG, cardiolipin and lysyl-PG) is used for building Gram-positive bacterial-membrane vesicles. Furthermore, we construct stable multi-compartment biomimicking vesicles using the gel-assisted swelling method. Importantly, we validate the bio-application of the bacterial vesicle models by quantifying diffusion of chemically synthetic amphoteric antibiotics. The transport rate is pH-responsive and depends on the lipid composition, based on which a permeation model is proposed. The permeability properties of antimicrobial peptides reveal pH dependent pore-forming activity in the model vesicles. Finally, we demonstrate the functionality of the vesicles by quantifying the uptake of membrane-impermeable molecules facilitated by embedded pore-forming proteins. We suggest that the bacterial vesicle models developed here can be used to understand fundamental biological processes like the peptide assembly mechanism or bacterial cell division and will have a multitude of applications in the bottom-up assembly of a protocell.

Giant vesicle functional models mimicking a bacterial membrane under physiological conditions are constructed.     

Selective single-atom electrocatalysts: a review with a focus on metal-doped covalent triazine frameworks

Single-atom electrocatalysts (SACs), which comprise singly isolated metal sites supported on heterogeneous substrates, have attracted considerable recent attention as next-generation electrocatalysts for various key reactions from the viewpoint of the environment and energy. Not only electrocatalytic activity but also selectivity can be precisely tuned via the construction of SACs with a defined coordination structure, such as homogeneous organometallics. Covalent organic frameworks (COFs) are promising supports for single-atom sites with designed coordination environments due to their unique physicochemical properties, which include porous structures, robustness, a wide range of possible designs, and abundant heteroatoms to coordinate single-metal sites. The rigid frameworks of COFs can hold unstable single-metal atoms, such as coordinatively unsaturated sites or easily aggregated Pt-group metals, which exhibit unique electrocatalytic selectivity. This minireview summarizes recent advances in the selective reactions catalysed by SACs, mainly those supported on triazine-based COFs.

Single-atom electrocatalysts (SACs) have attracted considerable attention as selective electrocatalysts. Metal-doped covalent triazine frameworks will be a novel platform for selective SACs to solve energy and environmental issues.     

Site-selective aqueous C–H acylation of tyrosine-containing oligopeptides with aldehydes

The development of useful synthetic tools to label amino acids within a peptide framework for the ultimate modification of proteins in a late-stage fashion is a challenging task of utmost importance within chemical biology. Herein, we report the first Pd-catalyzed C–H acylation of a collection of Tyr-containing peptides with aldehydes. This water-compatible tagging technique is distinguished by its site-specificity, scalability and full tolerance of sensitive functional groups. Remarkably, it provides straightforward access to a high number of oligopeptides with altered side-chain topology including mimetics of endomorphin-2 and neuromedin N, thus illustrating its promising perspectives toward the diversification of structurally complex peptides and chemical ligation.

A novel Pd-catalyzed C–H acylation reaction with readily available aldehydes under an aqueous environment towards the assembly of non-protegenic acylated Tyr-containing oligopeptides is presented.     

Expedient synthesis of conjugated triynes via alkyne metathesis

The first synthesis of conjugated triynes by molybdenum-catalysed alkyne metathesis is reported. Strategic to the success of this approach is the utilization of sterically-hindered diynes that allowed for the site-selective alkyne metathesis to produce the desired conjugated triyne products. The steric hindrance of the alkyne moiety was found to be crucial in preventing the formation of diyne byproducts. This novel synthetic strategy was amenable to self- and cross-metathesis providing straightforward access to the corresponding symmetrical and dissymmetrical triynes with high selectivity.

The first synthesis of symmetrical and dissymmetrical conjugated triynes by self- and cross-metathesis was successfully achieved thanks to the use of hindered diynes.     

Direct observation of ion emission from charged aqueous nanodrops: effects on gaseous macromolecular charging

Mechanistic information about how gaseous ions are formed from charged droplets has been difficult to establish because direct observation of nanodrops in a size range relevant to gaseous macromolecular ion formation by optical or traditional mass spectrometry methods is challenging owing to their small size and heterogeneity. Here, the mass and charge of individual aqueous nanodrops between 1–10 MDa (15–32 nm diameter) with ∼50–300 charges are dynamically monitored for 1 s using charge detection mass spectrometry. Discrete losses of minimally solvated singly charged ions occur, marking the first direct observation of ion emission from aqueous nanodrops in late stages of droplet evaporation relevant to macromolecular ion formation in native mass spectrometry. Nanodrop charge depends on the identity of constituent ions, with pure water nanodrops charged slightly above the Rayleigh limit and aqueous solutions containing alkali metal ions charged progressively below the Rayleigh limit with increasing cation size. MS2 capsid ions (∼3.5 MDa; ∼27 nm diameter) are more highly charged from aqueous ammonium acetate than from its biochemically preferred, 100 mM NaCl/10 mM Na phosphate solution, consistent with ion emission reducing the nanodrop and resulting capsid charge. The extent of charging indicates that the capsid partially collapses inside the nanodrops prior to the charging and formation of the dehydrated gaseous ions. These results demonstrate that ion emission can affect macromolecular charging and that conformational changes to macromolecular structure can occur in nanodrops prior to the formation of naked gaseous ions.

Ion evaporation from aqueous nanodrops is measured for the first time using charge detection mass spectrometry, and the origin of solute ion dependent charging of large (MDa) macromolecules is revealed.