Phosphinoborylenes as stable sources of fleeting borylenes

Base-stabilised borylenes that mimic the ability of transition metals to bind and activate inert substrates have attracted significant attention in recent years. However, such species are typically highly reactive and fleeting, and often cannot be isolated at ambient temperature. Herein, we describe a readily accessible trimethylphosphine-stabilised borylborylene which was found to possess a labile P–B bond that reversibly cleaves upon gentle heating. Exchange of the labile phosphine with other nucleophiles (CO, isocyanide, 4-dimethylaminopyridine) was investigated, and the binding strength of a range of potential borylene “ligands” has been evaluated computationally. The room-temperature-stable PMe₃-bound borylenes were subsequently applied to novel bond activations including [2 + 2] cycloaddition with carbodiimides and the reduction of dichalcogenides, revealing that PMe₃-stabilised borylenes can effectively behave as stable sources of the analogous fleeting dicoordinate species under mild conditions.

A room-temperature stable phosphinoborylene provides a source of a reactive two-coordinate borylene via dissociation of a labile phosphine upon gentle heating. Ligand exchange, the capture of unsaturated molecules, and oxidation have been explored.     

Synthetic biohybrid peptidoglycan oligomers enable pan-bacteria-specific labeling and imaging: in vitro and in vivo

Peptidoglycan is the core component of the bacterial cell wall, which makes it an attractive target for the development of bacterial targeting agents. Intercepting its enzymatic assembly with synthetic substrates allows for labeling and engineering of live bacterial cells. Over the past two decades, small-molecule-based labeling agents, such as antibiotics, d-amino acids or monosaccharides have been developed for probing biological processes in bacteria. Herein, peptidoglycan oligomers, substrates for transglycosylation, are prepared for the first time using a top-down approach, which starts from chitosan as a cheap feedstock. A high efficiency of labeling has been observed in all bacterial strains tested using micromolar substrates. In contrast, uptake into mammalian cells was barely observable. Additional mechanistic studies support a hypothesis of bacteria-specific metabolic labeling rather than non-specific binding to the bacterial surface. Eventually, its practicality in bacterial targeting capability is demonstrated in resistant strain detection and in vivo infection models.

Peptidoglycan oligomers have been derived from chitosan, using a top-down bio-hybrid strategy, as highly bacteria-specific substrates.     

Fluorogenic probes for detecting deacylase and demethylase activity towards post-translationally-modified lysine residues

Reversible enzymatic post-translational modification of the ε-amino groups of lysine residues (e.g. N-acylation reactions) plays an important role in regulating the cellular activities of numerous proteins. This study describes how enzyme catalyzed N-deprotection of lysine residues of non-fluorescent peptide-coumarin probes can be used to generate N-deprotected peptides that undergo spontaneous O- to N-ester transfer reactions (uncatalyzed) to generate a highly fluorescent N-carbamoyl peptide. This enables detection of enzyme catalyzed N-deacetylation, N-demalonylation, N-desuccinylation and N-demethylation reactions activities towards the N-modified lysine residues of these probes using simple ‘turn on’ fluorescent assays.

We developed “turn-on” fluorescent probes that detect enzymatic lysine deacylation and demethylation critical for epigenetic and other cellular phenomena, using intramolecular O- to N-ester transfer reactions.     

Proteomimetic surface fragments distinguish targets by function

The fragment-centric design promises a means to develop complex xenobiotic protein surface mimetics, but it is challenging to find locally biomimetic structures. To address this issue, foldameric local surface mimetic (LSM) libraries were constructed. Protein affinity patterns, ligand promiscuity and protein druggability were evaluated using pull-down data for targets with various interaction tendencies and levels of homology. LSM probes based on H14 helices exhibited sufficient binding affinities for the detection of both orthosteric and non-orthosteric spots, and overall binding tendencies correlated with the magnitude of the target interactome. Binding was driven by two proteinogenic side chains and LSM probes could distinguish structurally similar proteins with different functions, indicating limited promiscuity. Binding patterns displayed similar side chain enrichment values to those for native protein–protein interfaces implying locally biomimetic behavior. These analyses suggest that in a fragment-centric approach foldameric LSMs can serve as useful probes and building blocks for undruggable protein interfaces.

Foldameric local surface mimetics (LSMs) detect spots at protein surfaces and are promising building blocks in a fragment-centric design of xenobiotic structures and protein–protein interaction inhibitors.     

Catalytic asymmetric synthesis of N-substituted tetrahydroquinoxalines via regioselective Heyns rearrangement and stereoselective transfer hydrogenation in one pot

N-Substituted tetrahydroquinoxalines (37 examples) were step-economically obtained in good yield (<97%) and ee (<99%) with readily available substrates. The reaction proceeds through an interesting regioselective Heyns rearrangement/enantioselective transfer hydrogenation in one pot. The substrate scope and the reaction mechanism were systematically investigated.

N-Substituted tetrahydroquinoxalines were step-economically obtained in good yield and ee with readily available substrates.     

Target-templated de novo design of macrocyclic d-/l-peptides: discovery of drug-like inhibitors of PD-1

Peptides are a rapidly growing class of therapeutics with various advantages over traditional small molecules, especially for targeting difficult protein–protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing bioactive cyclic topologies that go beyond natural l-amino acids. Here, we report a generalizable framework that exploits the computational power of Rosetta, in terms of large-scale backbone sampling, side-chain composition and energy scoring, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we developed two new inhibitors (PD-i3 and PD-i6) of programmed cell death 1 (PD-1), a key immune checkpoint in oncology. A comprehensive biophysical evaluation was performed to assess their binding to PD-1 as well as their blocking effect on the endogenous PD-1/PD-L1 interaction. Finally, NMR elucidation of their in-solution structures confirmed our de novo design approach.

In silico design of heterochiral cyclic peptides that bind to a specific surface patch on the target protein (PD-1, in this case) and disrupt protein–protein interactions.     

Molecular mechanism of secreted amyloid-β precursor protein in binding and modulating GABABR1a

A recent phenomenal study discovered that the extension domain of secreted amyloid-β precursor protein (sAPP) can bind to the intrinsically disordered sushi 1 domain of the γ-aminobutyric acid type B receptor subunit 1a (GABA_BR1a) and modulate its synaptic transmission. The work provided an important structural foundation for the modulation of GABA_BR1a; however, the detailed molecular interaction mechanism, crucial for future drug design, remains elusive. Here, we further investigated the dynamical interactions between sAPP peptides and the natively unstructured sushi 1 domain using all-atom molecular dynamics simulations, for both the 17-residue sAPP peptide (APP 17-mer) and its minimally active 9 residue segment (APP 9-mer). We then explored mutations of the APP 9-mer with rigorous free energy perturbation (FEP) calculations. Our in silico mutagenesis studies revealed key residues (D4, W6, and W7) responsible for the binding with the sushi 1 domain. More importantly, one double mutation based on different vertebrate APP sequences from evolution exhibited a stronger binding (ΔΔG = −1.91 ± 0.66 kcal mol⁻¹), indicating a potentially enhanced GABA_BR1a modulator. These large-scale simulations may provide new insights into the binding mechanism between sAPP and the sushi 1 domain, which could open new avenues in the development of future GABA_BR1a-specific therapeutics.

A recent phenomenal study discovered that the extension domain of secreted amyloid-β precursor protein (sAPP) can bind to the intrinsically disordered sushi 1 domain of the γ-aminobutyric acid type B receptor subunit 1a (GABA_BR1a) and modulate its synaptic transmission.     

Uncovering the interactions driving carotenoid binding in light-harvesting complexes

Carotenoids are essential constituents of plant light-harvesting complexes (LHCs), being involved in protein stability, light harvesting, and photoprotection. Unlike chlorophylls, whose binding to LHCs is known to require coordination of the central magnesium, carotenoid binding relies on weaker intermolecular interactions (such as hydrogen bonds and van der Waals forces), whose character is far more elusive. Here we addressed the key interactions responsible for carotenoid binding to LHCs by combining molecular dynamics simulations and polarizable quantum mechanics/molecular mechanics calculations on the major LHC, LHCII. We found that carotenoid binding is mainly stabilized by van der Waals interactions with the surrounding chlorophyll macrocycles rather than by hydrogen bonds to the protein, the latter being more labile than predicted from structural data. Furthermore, the interaction network in the binding pockets is relatively insensitive to the chemical structure of the embedded carotenoid. Our results are consistent with a number of experimental data and challenge the role played by specific interactions in the assembly of pigment-protein complexes.

Carotenoids are essential constituents of plant light-harvesting complexes. This in silico study shows that carotenoid binding is mainly driven by van der Waals interactions with the surrounding chlorophylls rather than hydrogen bonds to the protein.     

Energetics of a protein disorder–order transition in small molecule recognition

Many proteins recognise other proteins via mechanisms that involve the folding of intrinsically disordered regions upon complex formation. Here we investigate how the selectivity of a drug-like small molecule arises from its modulation of a protein disorder-to-order transition. Binding of the compound AM-7209 has been reported to confer order upon an intrinsically disordered ‘lid’ region of the oncoprotein MDM2. Calorimetric measurements revealed that truncation of the lid region of MDM2 increases the apparent dissociation constant of AM-7209 250-fold. By contrast, lid truncation has little effect on the binding of the ligand Nutlin-3a. Insights into these differential binding energetics were obtained via a complete thermodynamic analysis that featured adaptive absolute alchemical free energy of binding calculations with enhanced-sampling molecular dynamics simulations. The simulations reveal that in apo MDM2 the ordered lid state is energetically disfavoured. AM-7209, but not Nutlin-3a, shows a significant energetic preference for ordered lid conformations, thus shifting the balance towards ordering of the lid in the AM-7209/MDM2 complex. The methodology reported herein should facilitate broader targeting of intrinsically disordered regions in medicinal chemistry.

Molecular simulations and biophysical measurements elucidate why the ligand AM-7209 orders a disordered region of the protein MDM2 on binding. This work expands strategies available to medicinal chemists for targeting disordered proteins.     

Small peptide diversification through photoredox-catalyzed oxidative C-terminal modification

A photoredox-catalyzed oxidative decarboxylative coupling of small peptides is reported, giving access to a variety of N,O-acetals. They were used as intermediates for the addition of phenols and indoles, leading to novel peptide scaffolds and bioconjugates. Amino acids with nucleophilic side chains, such as serine, threonine, tyrosine and tryptophan, could also be used as partners to access tri- and tetrapeptide derivatives with non-natural cross-linking.

A photoredox approach for the generation of N-acyliminiums derived from peptides enabling diversification via Friedel–Crafts reactions.     

Enhanced enzymatic activity exerted by a packed assembly of a single type of enzyme

In contrast to the dilute conditions employed for in vitro biochemical studies, enzymes are spatially organized at high density in cellular micro-compartments. In spite of being crucial for cellular functions, enzymatic reactions in such highly packed states have not been fully addressed. Here, we applied a protein adaptor to assemble a single type of monomeric enzyme on a DNA scaffold in the packed or dispersed states for carbonic anhydrase. The enzymatic reactions proceeded faster in the packed than in the dispersed state. Acceleration of the reaction in the packed assembly was more prominent for substrates with higher hydrophobicity. In addition, carbonic anhydrase is more tolerant of inhibitors in the packed assembly. Such an acceleration of the reaction in the packed state over the dispersed state was also observed for xylose reductase. We propose that the entropic force of water increases local substrate or cofactor concentration within the domain confined between enzyme surfaces, thus accelerating the reaction. Our system provides a reasonable model of enzymes in a packed state; this would help in engineering artificial metabolic systems.

The enzymatic reactions proceeded faster in the packed than in the dispersed state.     

Probe metal binding mode of imine covalent organic frameworks: cycloiridation for (photo)catalytic hydrogen evolution from formate

Metalation of covalent organic frameworks (COFs) is a critical strategy to functionalize COFs for advanced applications yet largely relies on the pre-installed specific metal docking sites in the network, such as porphyrin, salen, 2,2′-bipyridine, etc. We show in this study that the imine linkage of simple imine-based COFs, one of the most popular COFs, readily chelate transition metal (Ir in this work) via cyclometalation, which has not been explored before. The iridacycle decorated COF exhibited more than 10-fold efficiency enhancement in (photo)catalytic hydrogen evolution from aqueous formate solution than its molecular counterpart under mild conditions. This work will inspire more functional cyclometallated COFs to be explored beyond catalysis considering the large imine COF library and the rich metallacycle chemistry.

This study describes cyclometallation as a new metal binding mode for imine-based COFs. The iridacycle decorated COF could be used for catalytic hydrogen evolution from aqueous formate solution with high stability and high efficacy.     

Prebiotic access to enantioenriched glyceraldehyde mediated by peptides

A prebiotically plausible route to enantioenriched glyceraldehyde is reported via a kinetic resolution mediated by peptides. The reaction proceeds via a selective reaction between the l-peptide and the l-sugar producing an Amadori rearrangement byproduct and leaving d-glyceraldehyde in excess. Solubility considerations in the synthesis of proline–valine (pro–val) peptides allow nearly enantiopure pro–val to be formed starting from racemic pro and nearly racemic (10%) ee val. (ee = enantiomeric excess = (|d − l|)/(d + l)) Thus enantioenrichment of glyceraldehyde is achieved in a system with minimal initial chiral bias. This work demonstrates synergy between amino acids and sugars in the emergence of biological homochirality.

A prebiotically plausible route to enantioenriched glyceraldehyde is reported via a kinetic resolution mediated by peptides.     

Targeting structural features of viral genomes with a nano-sized supramolecular drug

RNA targeting is an exciting frontier for drug design. Intriguing targets include functional RNA structures in structurally-conserved untranslated regions (UTRs) of many lethal viruses. However, computational docking screens, valuable in protein structure targeting, fail for inherently flexible RNA. Herein we harness MD simulations with Markov state modeling to enable nanosize metallo-supramolecular cylinders to explore the dynamic RNA conformational landscape of HIV-1 TAR untranslated region RNA (representative for many viruses) replicating experimental observations. These cylinders are exciting as they have unprecedented nucleic acid binding and are the first supramolecular helicates shown to have anti-viral activity in cellulo: the approach developed in this study provides additional new insight about how such viral UTR structures might be targeted with the cylinder binding into the heart of an RNA-bulge cavity, how that reduces the conformational flexibility of the RNA and molecular details of the insertion mechanism. The approach and understanding developed represents a new roadmap for design of supramolecular drugs to target RNA structural motifs across biology and nucleic acid nanoscience.

MD simulations and Markov state modeling explore induced fit binding of metallo-helicates to bulges in dynamic TAR RNA, reproduce experimental data, show how RNA conformational flexibility is reduced, and give mechanistic insight into insertion.     

Metal–ligand bond strength determines the fate of organic ligands on the catalyst surface during the electrochemical CO2 reduction reaction

Colloidally synthesised nanocrystals (NCs) are increasingly utilised as catalysts to drive both thermal and electrocatalytic reactions. Their well-defined size and shape, controlled by organic ligands, are ideal to identify the parameters relevant to the activity, selectivity and stability in catalysis. However, the impact of the native surface ligands during catalysis still remains poorly understood, as does their fate. CuNCs are among the state-of-the-art catalysts for the electrochemical CO₂ reduction reaction (CO₂RR). In this work, we study CuNCs that are capped by different organic ligands to investigate their impact on the catalytic properties. We show that the latter desorb from the surface at a cathodic potential that depends on their binding strength with the metal surface, rather than their own electroreduction potentials. By monitoring the evolving surface chemistry in situ, we find that weakly bound ligands desorb very rapidly while strongly bound ligands impact the catalytic performance. This work provides a criterion to select labile ligands versus ligands that will persist on the surface, thus offering opportunity for interface design.

The metal–ligand binding strength is a key parameter in determining the role and fate of the surface ligands on nanoparticle catalysts during the electrochemical CO₂ reduction reaction.     

Catalyst design in C–H activation: a case study in the use of binding free energies to rationalise intramolecular directing group selectivity in iridium catalysis

Remote directing groups in a bifunctional molecule do not always behave independently of one another in C–H activation chemistries. A combined DFT and experimental mechanistic study to provide enhanced Ir catalysts for chemoselective C–H deuteration of bifunctional aryl primary sulfonamides is described. This provides a pharmaceutically-relevant and limiting case study in using binding energies to predict intramolecular directing group chemoselectivity. Rational catalyst design, guided solely by qualitative substrate–catalyst binding free energy predictions, enabled intramolecular discrimination between competing ortho-directing groups in C–H activation and delivered improved catalysts for sulfonamide-selective C–H deuteration. As a result, chemoselective binding of the primary sulfonamide moiety was achieved in the face of an intrinsically more powerful pyrazole directing group present in the same molecule. Detailed DFT calculations and mechanistic experiments revealed a breakdown in the applied binding free energy model, illustrating the important interconnectivity of ligand design, substrate geometry, directing group cooperativity, and solvation in supporting DFT calculations. This work has important implications around attempts to predict intramolecular C–H activation directing group chemoselectivity using simplified monofunctional fragment molecules. More generally, these studies provide insights for catalyst design methods in late-stage C–H functionalisation.

In C–H activation chemistries, the interpretation of the influence of remote directing groups in a bifunctional molecule depends on the in silico method used to inform catalyst design.     

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.     

Direct,stereoselective thioglycosylation enabled by an organophotoredox radical strategy

While strategies involving a 2e⁻ transfer pathway have dictated glycosylation development, the direct glycosylation of readily accessible glycosyl donors as radical precursors is particularly appealing because of high radical anomeric selectivity and atom- and step-economy. However, the development of the radical process has been challenging owing to notorious competing reduction, elimination and/or S_N side reactions of commonly used, labile glycosyl donors. Here we introduce an organophotocatalytic strategy through which glycosyl bromides can be efficiently converted into corresponding anomeric radicals by photoredox mediated HAT catalysis without a transition metal or a directing group and achieve highly anomeric selectivity. The power of this platform has been demonstrated by the mild reaction conditions enabling the synthesis of challenging α-1,2-cis-thioglycosides, the tolerance of various functional groups and the broad substrate scope for both common pentoses and hexoses. Furthermore, this general approach is compatible with both sp² and sp³ sulfur electrophiles and late-stage glycodiversification for a total of 50 substrates probed.

Organophotoredox mediated HAT catalysis is developed for achieving high anomerically selective thioglycosylation of glycosyl bromides.     

A FRET-based fluorescent Zn2+ sensor: 3D ratiometric imaging,flow cytometric tracking and cisplatin-induced Zn2+ fluctuation monitoring

Monitoring labile Zn²⁺ homeostasis is of great importance for the study of physiological functions of Zn²⁺ in biological systems. Here we report a novel ratiometric fluorescent Zn²⁺ sensor, CPBT, which was constructed based on chelation-induced alteration of FRET efficiency. CPBT was readily cell membrane permeable and showed a slight preferential localization in the endoplasmic reticulum. With this sensor, 3D ratiometric Zn²⁺ imaging was first realized in the head of zebra fish larvae via Z-stack mode. CPBT could track labile Zn²⁺ in a large number of cells through ratiometric flow cytometric assay. More interestingly, both ratiometric fluorescence imaging and flow cytometric assay demonstrated that the labile Zn²⁺ level in MCF-7 cells (cisplatin-sensitive) decreased while that in SKOV3 cells (cisplatin-insensitive) increased after cisplatin treatment, indicating that Zn²⁺ may play an important role in cisplatin induced signaling pathways in these cancer cells.

A Zn²⁺ sensor exhibiting 3D ratiometric imaging and flow cytometric ability was constructed based on the FRET mechanism, and cisplatin-induced endogenous labile Zn²⁺ fluctuations were monitored in real time.     

Computational strategy for intrinsically disordered protein ligand design leads to the discovery of p53 transactivation domain I binding compounds that activate the p53 pathway

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.

A hierarchical computational strategy for IDP drug virtual screening (IDPDVS) was proposed and successfully applied to identify compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells.