A new pentacyclic pyrylium fluorescent probe that responds to pH imbalance during apoptosis

Efficient fluorophores with easy synthetic routes and fast responses are of great importance in clinical diagnostics. Herein, we report a new, rigid pentacyclic pyrylium fluorophore, PS-OMe, synthesised in a single step by a modified Vilsmeier–Haack reaction. Insights into the reaction mechanism facilitated a new reaction protocol for the efficient synthesis of PS-OMe which upon demethylation resulted in a “turn-on” pH sensor, PS-OH. This new fluorescent probe has been successfully used to monitor intracellular acidification at physiological pH. From the fluorescence image analysis, we were able to quantify the intracellular dynamic pH change during apoptosis. This new pH probe is a potential chemical tool for screening, drug discovery and dose determination in cancer therapy.

A modified Vilsmeier–Haack reaction resulted in the synthesis of a pyrylium based turn-on fluorescent pH probe. The probe can monitor minute acidification and dynamic pH variation in cells during apoptosis with therapeutic chemo drugs.     

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Impermeable SNAP-tag substrates allow exclusive labelling of receptors on the cell membrane for nanoscopy, SiMPull and in vivo use.     

Generation of oligonucleotide conjugates via one-pot diselenide-selenoester ligation–deselenization/alkylation

A breadth of strategies are needed to efficiently modify oligonucleotides with peptides or lipids to capitalize on their therapeutic and diagnostic potential, including the modulation of in vivo chemical stability and for applications in cell-targeting and cell-permeability. The chemical linkages typically used in peptide oligonucleotide conjugates (POCs) have limitations in terms of stability and/or ease of synthesis. Herein, we report an efficient method for POC synthesis using a diselenide-selenoester ligation (DSL)-deselenization strategy that rapidly generates a stable amide linkage between the two biomolecules. This conjugation strategy is underpinned by a novel selenide phosphoramidite building block that can be incorporated into an oligonucleotide by solid-phase synthesis to generate diselenide dimer molecules. These can be rapidly ligated with peptide selenoesters and, following in situ deselenization, lead to the efficient generation of POCs. The diselenide within the oligonucleotide also serves as a flexible functionalisation handle that can be leveraged for fluorescent labelling, as well as for alkylation to generate micelles.

An efficient and versatile approach for the late-stage generation of oligonucleotide conjugates by diselenide-selenoester ligation (DSL)–deselenization/alkylation was developed.     

Mechanistic insights into copper-catalyzed aerobic oxidative coupling of N–N bonds

Catalytic N–N coupling is a valuable transformation for chemical synthesis and energy conversion. Here, mechanistic studies are presented for two related copper-catalyzed oxidative aerobic N–N coupling reactions, one involving the synthesis of a pharmaceutically relevant triazole and the other relevant to the oxidative conversion of ammonia to hydrazine. Analysis of catalytic and stoichiometric N–N coupling reactions support an “oxidase”-type catalytic mechanism with two redox half-reactions: (1) aerobic oxidation of a Cu^I catalyst and (2) Cu^II-promoted N–N coupling. Both reactions feature turnover-limiting oxidation of Cu^I by O₂, and this step is inhibited by the N–H substrate(s). The results highlight the unexpected facility of the N–N coupling step and establish a foundation for development of improved catalysts for these transformations.

Mechanistic studies provide valuable insights into Cu-catalyzed N–N coupling reactions relevant to energy conversion and pharmaceutical synthesis.     

Single point activation of pyridines enables reductive hydroxymethylation

The single point activation of pyridines, using an electron-deficient benzyl group, facilitates the ruthenium-catalysed dearomative functionalisation of a range of electronically diverse pyridine derivatives. This transformation delivers hydroxymethylated piperidines in good yields, allowing rapid access to medicinally relevant small heterocycles. A noteworthy feature of this work is that paraformaldehyde acts as both a hydride donor and an electrophile in the reaction, enabling the use of cheap and readily available feedstock chemicals. Removal of the activating group can be achieved readily, furnishing the free NH compound in only 2 steps. The synthetic utility of the method was illustrated with a synthesis of (±)-Paroxetine.

Pyridines can be activated at a single point with a new benzyl group, followed by dearomative functionalisation at C3 using formaldehyde.     

Pinkment: a synthetic platform for the development of fluorescent probes for diagnostic and theranostic applications

Reaction-based fluorescent-probes have proven successful for the visualisation of biological species in various cellular processes. Unfortunately, in order to tailor the design of a fluorescent probe to a specific application (i.e. organelle targeting, material and theranostic applications) often requires extensive synthetic efforts and the synthetic screening of a range of fluorophores to match the required synthetic needs. In this work, we have identified Pinkment-OH as a unique “plug-and-play” synthetic platform that can be used to develop a range of ONOO⁻ responsive fluorescent probes for a variety of applications. These include theranostic-based applications and potential material-based/bioconjugation applications. The as prepared probes displayed an excellent sensitivity and selectivity for ONOO⁻ over other ROS. In vitro studies using HeLa cells and RAW 264.7 macrophages demonstrated their ability to detect exogenously and endogenously produced ONOO⁻. Evaluation in an LPS-induced inflammation mouse model illustrated the ability to monitor ONOO⁻ production in acute inflammation. Lastly, theranostic-based probes enabled the simultaneous evaluation of indomethacin-based therapeutic effects combined with the visualisation of an inflammation biomarker in RAW 264.7 cells.

Pinkment, a resorufin based ONOO⁻ selective and sensitive ‘plug and play’ fluorescence-based platform for in vitro and in vivo use, enables facile functionalisation for various imaging and theranostic applications.     

Understanding the geometric diversity of inorganic and hybrid frameworks through structural coarse-graining

Much of our understanding of complex structures is based on simplification: for example, metal–organic frameworks are often discussed in the context of “nodes” and “linkers”, allowing for a qualitative comparison with simpler inorganic structures. Here we show how such an understanding can be obtained in a systematic and quantitative framework, combining atom-density based similarity (kernel) functions and unsupervised machine learning with the long-standing idea of “coarse-graining” atomic structure. We demonstrate how the latter enables a comparison of vastly different chemical systems, and we use it to create a unified, two-dimensional structure map of experimentally known tetrahedral AB₂ networks – including clathrate hydrates, zeolitic imidazolate frameworks (ZIFs), and diverse inorganic phases. The structural relationships that emerge can then be linked to microscopic properties of interest, which we exemplify for structural heterogeneity and tetrahedral density.

A coarse-graining approach enables structural comparisons across vastly different chemical spaces, from inorganic polymorphs to hybrid framework materials.     

Towards photoswitchable quadruple hydrogen bonds via a reversible “photolocking” strategy for photocontrolled self-assembly

Developing new photoswitchable noncovalent interaction motifs with controllable bonding affinity is crucial for the construction of photoresponsive supramolecular systems and materials. Here we describe a unique “photolocking” strategy for realizing photoswitchable control of quadruple hydrogen-bonding interactions on the basis of modifying the ureidopyrimidinone (UPy) module with an ortho-ester substituted azobenzene unit as the “photo-lock”. Upon light irradiation, the obtained Azo-UPy motif is capable of unlocking/locking the partial H-bonding sites of the UPy unit, leading to photoswitching between homo- and heteroquadruple hydrogen-bonded dimers, which has been further applied for the fabrication of novel tunable hydrogen bonded supramolecular systems. This “photolocking” strategy appears to be broadly applicable in the rational design and construction of other H-bonding motifs with sufficiently photoswitchable noncovalent interactions.

A photolocking strategy is described to achieve the construction of effectively photoswitchable quadruple hydrogen bonds featuring with photoregulable H-bonding affinities, which is further applied in the photocontrollable H-bonded self-assemblies.     

meta-Selective C–H functionalisation of aryl boronic acids directed by a MIDA-derived boronate ester

N-Methyliminodiacetic acid (MIDA) boronates are boronic acid derivatives which are stable to reduction, oxidation and transmetalation. This has led to their widespread use as boronic acid protecting groups (PGs) and in iterative cross-couplings. We describe herein the development of a novel MIDA derivative that acts in a dual manner, as a protecting group and a directing group (DG) for meta C(sp²)–H functionalisation of arylboronic acids. Palladium catalysed C–H alkenylations, acetoxylations and arylations are possible, at room temperature and under aerobic conditions. Deprotection to reveal the functionalised boronic acids is rapid and allows for full recovery of the DG. The technique allows the facile diversification of aryl boronic acids and their subsequent use in a range of reactions or in iterative processes.

An N-methyliminodiacetic acid derivative allows the meta-C–H functionalisation of boronic acids, acting simultaneously as a directing and protecting group.     

Chemical tuning of spin clock transitions in molecular monomers based on nuclear spin-free Ni(ii)

We report the existence of a sizeable quantum tunnelling splitting between the two lowest electronic spin levels of mononuclear Ni complexes. The level anti-crossing, or magnetic “clock transition”, associated with this gap has been directly monitored by heat capacity experiments. The comparison of these results with those obtained for a Co derivative, for which tunnelling is forbidden by symmetry, shows that the clock transition leads to an effective suppression of intermolecular spin–spin interactions. In addition, we show that the quantum tunnelling splitting admits a chemical tuning via the modification of the ligand shell that determines the crystal field and the magnetic anisotropy. These properties are crucial to realize model spin qubits that combine the necessary resilience against decoherence, a proper interfacing with other qubits and with the control circuitry and the ability to initialize them by cooling.

We have directly monitored spin level anti-crossings, or “clock transitions”, in Ni(ii) molecular monomers and shown that the quantum tunnelling gap admits a chemical tuning.     

Rational design of an “all-in-one” phototheranostic

We report here porphodilactol derivatives and their corresponding metal complexes. These systems show promise as “all-in-one” phototheranostics and are predicated on a design strategy that involves controlling the relationship between intersystem crossing (ISC) and photothermal conversion efficiency following photoexcitation. The requisite balance was achieved by tuning the aromaticity of these porphyrinoid derivatives and forming complexes with one of two lanthanide cations, namely Gd³⁺ and Lu³⁺. The net result led to a metalloporphodilactol system, Gd-trans-2, with seemingly optimal ISC efficiency, photothermal conversion efficiency and fluorescence properties, as well as good chemical stability. Encapsulation of Gd-trans-2 within mesoporous silica nanoparticles (MSN) allowed its evaluation for tumour diagnosis and therapy. It was found to be effective as an “all-in-one” phototheranostic that allowed for NIR fluorescence/photoacoustic dual-modal imaging while providing an excellent combined PTT/PDT therapeutic efficacy in vitro and in vivo in 4T1-tumour-bearing mice.

We report here porphodilactol derivatives and their corresponding metal complexes as “all-in-one” phototheranostics by controlling the relationship between intersystem crossing (ISC) and photothermal conversion efficiency following photoexcitation.     

Catalytic C–H to C–M (M = Al,Mg) bond transformations with heterometallic complexes

C–H functionalisation is one of the cornerstones of modern catalysis and remains a topic of contemporary interest due its high efficiency and atom-economy. Among these reactions, C–H borylation, that is the transformation of C–H to C–B bonds, has experienced a fast development because of the wide utility of organoboron reagents as synthetic intermediates. The mechanistic background is now well-understood and the role of transition metal boryl or σ-borane intermediates in this transformation is well documented. This mini-review focuses on efforts made by our group, and others, to establish palladium- and calcium-catalysed methods for C–H metalation employing heavier main group elements (M = Al, Mg). These are new catalytic reactions first accomplished in our group that we have termed C–H alumination and magnesiation respectively. Unusual heterometallic complexes have been identified as key on-cycle intermediates and their unique reactivity is discussed in the context of new catalytic pathways for C–H functionalisation. Hence, this mini-review summarises the recent progress in the area of C–H metalation reactions as well as the new opportunities that may arise from this concept.

This highlight focuses on recent efforts to establish catalytic methods for C–H functionalisation with main group metals (M = Al, Mg).     

Attention-based generative models for de novo molecular design

Attention mechanisms have led to many breakthroughs in sequential data modeling but have yet to be incorporated into any generative algorithms for molecular design. Here we explore the impact of adding self-attention layers to generative β-VAE models and show that those with attention are able to learn a complex “molecular grammar” while improving performance on downstream tasks such as accurately sampling from the latent space (“model memory”) or exploring novel chemistries not present in the training data. There is a notable relationship between a model''s architecture, the structure of its latent memory and its performance during inference. We demonstrate that there is an unavoidable tradeoff between model exploration and validity that is a function of the complexity of the latent memory. However, novel sampling schemes may be used that optimize this tradeoff. We anticipate that attention will play an important role in future molecular design algorithms that can make efficient use of the detailed molecular substructures learned by the transformer.

An implementation of attention within the variational autoencoder framework for continuous representation of molecules. The addition of attention significantly increases model performance for complex tasks such as exploration of novel chemistries.     

Evaluation of fully-functionalized diazirine tags for chemical proteomic applications

The use of photo-affinity reagents for the mapping of noncovalent small molecule–protein interactions has become widespread. Recently, several ‘fully-functionalized’ (FF) chemical tags have been developed wherein a photoactivatable capture group, an enrichment handle, and a functional group for synthetic conjugation to a molecule of interest are integrated into a single modular tag. Diazirine-based FF tags in particular are increasingly employed in chemical proteomic investigations; however, despite routine usage, their relative utility has not been established. Here, we systematically evaluate several diazirine-containing FF tags, including a terminal diazirine analog developed herein, for chemical proteomic investigations. Specifically, we compared the general reactivity of five diazirine tags and assessed their impact on the profiles of various small molecules, including fragments and known inhibitors revealing that such tags can have profound effects on the proteomic profiles of chemical probes. Our findings should be informative for chemical probe design, photo-affinity reagent development, and chemical proteomic investigations.

The chemical proteomic properties of five diazirine-based, fully-functionalized photoaffinity tags, including a newly developed, minimal tag, were compared. This study provides guidance for the development of new photoaffinity probes.     

Thermal effects – an alternative mechanism for plasmon-assisted photocatalysis

Recent experiments claimed that the catalysis of reaction rates in numerous bond-dissociation reactions occurs via the decrease of activation barriers driven by non-equilibrium (“hot”) electrons in illuminated plasmonic metal nanoparticles. Thus, these experiments identify plasmon-assisted photocatalysis as a promising path for enhancing the efficiency of various chemical reactions. Here, we argue that what appears to be photocatalysis is much more likely thermo-catalysis, driven by the well-known plasmon-enhanced ability of illuminated metallic nanoparticles to serve as heat sources. Specifically, we point to some of the most important papers in the field, and show that a simple theory of illumination-induced heating can explain the extracted experimental data to remarkable agreement, with minimal to no fit parameters. We further show that any small temperature difference between the photocatalysis experiment and a control experiment performed under external heating is effectively amplified by the exponential sensitivity of the reaction, and is very likely to be interpreted incorrectly as “hot” electron effects.

A simple Arrhenius-based theory of heating, rather than “hot electrons”, can reproduce some high-profile photocatalysis experimental results to remarkable accuracy. Flaws in temperature measurement may have led to wrong conclusions.     

Structure and redox tuning of gas adsorption properties in calixarene-supported Fe(ii)-based porous cages

We describe the synthesis of Fe(ii)-based octahedral coordination cages supported by calixarene capping ligands. The most porous of these molecular cages has an argon accessible BET surface area of 898 m² g⁻¹ (1497 m² g⁻¹ Langmuir). The modular synthesis of molecular cages allows for straightforward substitution of both the bridging carboxylic acid ligands and the calixarene caps to tune material properties. In this context, the adsorption enthalpies of C₂/C₃ hydrocarbons ranged from −24 to −46 kJ mol⁻¹ at low coverage, where facile structural modifications substantially influence hydrocarbon uptakes. These materials exhibit remarkable stability toward oxidation or decomposition in the presence of air and moisture, but application of a suitable chemical oxidant generates oxidized cages over a controlled range of redox states. This provides an additional handle for tuning the porosity and stability of the Fe cages.

We describe the synthesis of Fe(ii)-based coordination cages whose stability and gas adsorption properties can be tuned through structural modifications and redox reactivity.     

A protecting group strategy to access stable lacunary polyoxomolybdates for introducing multinuclear metal clusters

Although metal-containing polyoxomolybdates (molybdenum oxide clusters) exhibit outstanding catalytic properties, their precise synthetic method has not yet been developed. This is mainly because the very low stability of the multivacant lacunary polyoxomolybdates limited their use as synthetic precursors. Here, we present a “protecting group strategy” in polyoxometalate synthesis and successfully develop an efficient method for synthesising multinuclear metal-containing polyoxomolybdates using pyridine as a protecting group for unstable trivacant lacunary Keggin-type polyoxomolybdate [PMo₉O₃₄]⁹⁻. Specifically, tetranuclear cubane- and planar-type manganese clusters were selectively synthesised in the polyoxomolybdates using the present method. The importance of this work is that, in addition to being the first practical way of utilizing multivacant lacunary polyoxomolybdates as precursors, this new “protecting group strategy” will make it possible to produce polyoxometalates with unexplored structures and properties.

A “protecting group strategy” for unstable lacunary polyoxomolybdates enabled successful synthesis of two types of tetranuclear manganese clusters.     

Covalent cell surface recruitment of chemotherapeutic polymers enhances selectivity and activity

Synthetic macromolecular chemotherapeutics inspired by host defence peptides can disrupt cell membranes and are emerging as agents for the treatment of cancer and infections. However, their off-target effects remain a major unmet challenge. Here we introduce a covalent recruitment strategy, whereby metabolic oligosaccharide engineering is used to label targeted cells with azido glycans, to subsequently capture chemotherapeutic polymers by a bio-orthogonal click reaction. This results in up to 10-fold reduction in EC₅₀ and widening of the therapeutic window. Cell death is induced by not only membrane leakage, but also by apoptosis due to the conjugated chemotherapeutic being internalised by glycan recycling. Covalent recruitment also lead to increased penetration and significant cell death in a 3-D tumour model in just 3 hours, whereas doxorubicin required 24 hours. This conceptual approach of ‘engineering cells to capture polymers’ rather than ‘engineering polymers to target cells’ will bring new opportunities in non-traditional macromolecular therapeutics.

Chemotherapeutic polymers are targeted to cells by introduction of unnatural glycans to their glycocalyx, enhancing their cytotoxic effect.