Fixing the Conformation of Calix[4]arenes: When Are Three Carbons Not Enough?

Calix[4]arenes are unique macrocycles that through judicious functionalisation at the lower rim can be either fixed in one of four conformations or remain conformationally flexible. Introduction of propynyl or propenyl groups unexpectedly provides a new possibility; a unidirectional conformational switch, with the 1,3-alternate and 1,2-alternate conformers switching to the partial cone conformation, whilst the cone conformation is unchanged, under standard experimental conditions. Using 1 H NMR kinetic studies, rates of switching have been shown to be dependent on the starting conformation, upper-rim substituent, where reduction in bulk enables faster switching, solvent and temperature with 1,2-alternate conformations switching fastest. Ab initio calculations (DFT) confirmed the relative stabilities of the conformations and point towards the partial cone conformer being the most stable of the four. The potential impact on synthesis through the "click" reaction has been investigated and found not to be significant.

Identification of new DNA i-motif binding ligands through a fluorescent intercalator displacement assay.

i-Motifs are quadruplex DNA structures formed from sequences rich in cytosine and held together by intercalated, hemi-protonated cytosine-cytosine base pairs. These sequences are prevalent in gene promoter regions and may play a role in gene transcription. Targeting these structures with ligands could provide a novel way to target genetic disease but there are very few ligands which have been shown to interact with i-motif DNA. Fluorescent intercalator displacement (FID) assays are a simple way to screen ligands against DNA secondary structures. Here we characterise how thiazole orange interacts with i-motif DNA and assess its ability for use in a FID assay. Additionally, we report FID-based ligand screening using thiazole orange against the i-motif forming sequence from the human telomere to reveal new i-motif binding compounds which have the potential for further development.

Pillar[5]arene-Based Glycoclusters: Synthesis and Multivalent Binding to Pathogenic Bacterial Lectins.

The synthesis of pillar[5]arene-based glycoclusters has been readily achieved by CuAAC conjugations of azido- and alkyne-functionalized precursors. The lectin binding properties of the resulting glycosylated multivalent ligands have been studied by at least two complementary techniques to provide a good understanding. Three lectins were selected from bacterial pathogens based on their potential therapeutic applications as anti-adhesives, namely LecA and LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria. As a general trend, multivalency improved the binding to lectins and a higher affinity can be obtained by increasing to a certain limit the length of the spacer arm between the carbohydrate subunits and the central macrocyclic core.

"Janus" Calixarenes: Double-Sided Molecular Linkers for Facile, Multianchor Point, Multifunctional, Surface Modification.

We herein report the synthesis of novel "Janus" calix[4]arenes bearing four "molecular tethering" functional groups on either the upper or lower rims of the calixarene. These enable facile multipoint covalent attachment to electrode surfaces with monolayer coverage. The other rim of the calixarenes bear either four azide or four ethynyl functional groups, which are easily modified by the copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), either pre- or postsurface modification, enabling these conical, nanocavity reactor sites to be decorated with a wide range of substrates to impart desired chemical properties. Redox active species decorating the peripheral rim are shown to be electrically connected by the calixarene to the electrode surface in either "up" or "down" orientations of the calixarene.

Open-resorcinarenes, a new family of multivalent scaffolds.

A new family of multivalent ligand platforms, the open-resorcinarenes, has been prepared in a straightforward two-step reaction. Modification of the core gives a range of topologically diverse scaffolds; functionalisation confirms the versatility of this approach, as shown through the formation of an octacalixarene array.

Pentavalent pillar[5]arene-based glycoclusters and their multivalent binding to pathogenic bacterial lectins.

Anti-adhesive glycoclusters offer potential as therapeutic alternatives to classical antibiotics in treating infections. Pillar[5]arenes functionalised with either five galactose or five fucose residues were readily prepared using CuAAC reactions and evaluated for their binding to three therapeutically relevant bacterial lectins: LecA and Lec B from Pseudomonas aeuruginosa and BambL from Burkholderia ambifaria. Steric interactions were demonstrated to be a key factor in achieving good binding to LecA with more flexible galactose glycoclusters showing enhanced activity. In contrast binding to the fucose-selective lectins confirmed the importance of topology of the glycoclusters for activity with the pillar[5]arene ligand proving a selective ligand for BambL.

Topological effects and binding modes operating with multivalent iminosugar-based glycoclusters and mannosidases.

Multivalent iminosugars have been recently explored for glycosidase inhibition. Affinity enhancements due to multivalency have been reported for specific targets, which are particularly appealing when a gain in enzyme selectivity is achieved but raise the question of the binding mode operating with this new class of inhibitors. Here we describe the development of a set of tetra- and octavalent iminosugar probes with specific topologies and an assessment of their binding affinities toward a panel of glycosidases including the Jack Bean α-mannosidase (JBαMan) and the biologically relevant class II α-mannosidases from Drosophila melanogaster belonging to glycohydrolase family 38, namely Golgi α-mannosidase ManIIb (GM) and lysosomal α-mannosidase LManII (LM). Very different inhibitory profiles were observed for compounds with identical valencies, indicating that the spatial distribution of the iminosugars is critical to fine-tune the enzymatic inhibitory activity. Compared to the monovalent reference, the best multivalent compound showed a dramatic 800-fold improvement in the inhibitory potency for JBαMan, which is outstanding for just a tetravalent ligand. The compound was also shown to increase both the inhibitory activity and the selectivity for GM over LM. This suggests that multivalency could be an alternative strategy in developing therapeutic GM inhibitors not affecting the lysosomal mannosidases. Dynamic light scattering experiments and atomic force microscopy performed with coincubated solutions of the compounds with JBαMan shed light on the multivalent binding mode. The multivalent compounds were shown to promote the formation of JBαMan aggregates with different sizes and shapes. The dimeric nature of the JBαMan allows such intermolecular cross-linking mechanisms to occur.

Rational design and synthesis of optimized glycoclusters for multivalent lectin-carbohydrate interactions: influence of the linker arm.

The design of multivalent glycoclusters requires the conjugation of biologically relevant carbohydrate epitopes functionalized with linker arms to multivalent core scaffolds. The multigram-scale syntheses of three structurally modified triethyleneglycol analogues that incorporate amide moiety(ies) and/or a phenyl ring offer convenient access to a series of carbohydrate probes with different water solubilities and rigidities. Evaluation of flexibility and determination of preferred conformations were performed by conformational analysis. Conjugation of the azido-functionalized carbohydrates with tetra-propargylated core scaffolds afforded a library of 18 tetravalent glycoclusters, in high yields, by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The compounds were evaluated for their ability to bind to PA-IL (the LecA lectin from the opportunistic pathogen Pseudomonas aeruginosa). Biochemical evaluation through inhibition of hemagglutination assays (HIA), enzyme-linked lectin assays (ELLA), surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) revealed improved and unprecedented affinities for one of the monovalent probes (K(d)=5.8 µM) and also for a number of the tetravalent compounds that provide several new nanomolar ligands for this tetrameric lectin.

Stable and sensitive probes for lysosomes: cell-penetrating fluorescent calix[4]arenes accumulate in acidic vesicles.

The uptake of a fluorescently labeled cationic calix[4] (NBDCalAm) in live, nonfixed cells has been investigated. The compound is taken into the cells rapidly and shows distinct endosomal distribution after 2 hours. This distribution pattern shows colocalization with lysosomal staining. The uptake is not altered by inhibition of clathrin or caveolae dependent pathways nor by depletion of the cellular ATP-pool. Immediately after uptake the probe is localized in the Golgi and brefeldin A treatment prevents transport to lysosomes. Pulse chase experiments with bafilomycin A1, monensin, and sodium azide showed that accumulation and retention of the probe in lysosomes is primarily driven by the activity of vacuolar ATPases. The NBD labeled calix[4]arene provides a very stable and sensitive marker for lysosomes, and has a considerable advantage over some commercially available lysosomal markers in so far that the fluorescent signal is stable even when the cells are incubated in dye-free medium after staining.

Synthesis and antimicrobial activity of brominated resorcinol dimers.

Dibrominated resorcinol dimers were synthesized by reaction of 4-bromoresorcinol with aldehydes under reflux in ethanol in the presence of HCl. Subsequent dehalogenation yielded the corresponding monobrominated compounds and a fully dehalogenated dimer. Of the dimers, 6,6'-((4-hydroxyphenyl)methylene)bis(4-bromobenzene-1,3-diol) (4) displayed potent antibacterial activity and inhibitory activity against isocitrate lyase Candida albicans.

CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins.

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.

29th Annual GP2A Medicinal Chemistry Conference.

The 29th Annual GP2A (Group for the Promotion of Pharmaceutical chemistry in Academia) Conference was a virtual event this year due to the COVID-19 pandemic and spanned three days from Wednesday 25 to Friday 27 August 2021. The meeting brought together an international delegation of researchers with interests in medicinal chemistry and interfacing disciplines. Abstracts of keynote lectures given by the 10 invited speakers, along with those of the 8 young researcher talks and the 50 flash presentation posters, are included in this report. Like previous editions, the conference was a real success, with high-level scientific discussions on cutting-edge advances in the fields of pharmaceutical chemistry.

Achieving high affinity towards a bacterial lectin through multivalent topological isomers of calix[4]arene glycoconjugates.

A family of seven topologically isomeric calix[4]arene glycoconjugates was prepared through the synthesis of a series of alkyne-derivatised calix[4]arene precursors that are suitable for the attachment of sugar moieties by microwave-assisted copper(I)-catalysed azide-alkyne cycloaddition (CuAAC). The glycoconjugates thus synthesised comprised one mono-functionalised derivative, two 1,2- or 1,3-divalent regioisomers, one trivalent and three tetravalent topoisomers in the cone, partial cone or 1,3-alternate conformations. The designed glycoconjugates were evaluated as ligands for the galactose-binding lectin PA-IL from the opportunistic bacterium Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. Binding affinities were determined by isothermal titration calorimetry (ITC), and the interaction with the lectin was shown to be strongly dependant on both the valence and the topology. Whereas the trivalent conjugate displayed enhanced affinity when compared to a monosaccharide model, the tetravalent conjugates are to-date the highest-affinity ligands measured by ITC. The topologies presenting carbohydrates on both faces of calixarene are the most potent ones with dissociation constants of approximately 200 nM. Molecular modelling suggests that such a multivalent molecule can efficiently chelate two of the binding sites of the tetrameric lectin; this explains the 800-fold increase of affinity achieved by the tetravalent molecule. Surface plasmon resonance (SPR) experiments confirmed that this glycoconjugate is the strongest inhibitor for binding of PA-IL to galactosylated surfaces for potential applications as an anti-adhesive agent.

A pyrenyl-appended triazole-based calix[4]arene as a fluorescent sensor for Cd2+ and Zn2+.

The synthesis and evaluation of a novel calix[4]arene-based fluorescent chemosensor 8 for the detection of Cd(2+) and Zn(2+) is described. The fluorescent spectra changes observed upon addition of various metal ions show that 8 is highly selective for Cd(2+) and Zn(2+) over other metal ions. Addition of Cd(2+) and Zn(2+) to the solution of 8 results in ratiometric measurement.

Calixarene alpha-ketoacetylenes: versatile platforms for reaction with hydrazine nucleophile.

Late stage diversification of calix[4]arenes and thiacalix[4]arenes with heterocycles remains a significant synthetic challenge and hampers further exploitation of the scaffolds. Here we describe the development of a short and facile synthetic route to conformationally diverse novel calix[4]arene and thiacalix[4]arene ynones using a palladium cross coupling approach (5% Pd(ii) + 10% Cu(i)) with benzoyl chloride. Their successful conversion to heterocycles to afford pyrazoles was demonstrated through treatment with hydrazine. Functionalisation is calixarene conformation and linker independent enabling access to a library of structures.

Efficient gene transfection with functionalised multicalixarenes.

Novel amino-functionalised multicalixarenes have been synthesised which show low cellular toxicity, effective DNA binding and, when featuring aliphatic amines, are efficient gene transfection agents.

25th Conference of GP2A.

The 25th Conference of GP2A was held on 31 August and 1 September 2017 in Liverpool, UK, with the aim of exchange of ideas and experience, particularly amongst young medicinal chemists. Topics included bioactive compounds from plants and lichens, and design and development of drugs. Abstracts of invited lectures, proffered oral presentations, flash presentations and posters presented during the meeting are collected in this report.

Antiadhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalized patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multiresistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells, and destruction of alveolar tissues were performed. The antiadhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

Encapsulation of drug molecules into calix[n]arene nanobaskets. role of aminocalix[n]arenes in biopharmaceutical field.

Calix[n]arenes (CAs) are supramolecular compounds able to form guest-host inclusion complexes with metal ions, small organic molecules, and small moieties of larger molecules. Although the CA literature is extensive, relatively few publications deal with water-soluble CAs, especially those containing nitrogen-based functionality. These CAs possess antibacterial and antifungal activity. Because of their molecular structure, they are surface active and also able to form water-soluble drug complexes, giving additional potential as enabling pharmaceutical excipients. This article provides an overview of the published data regarding synthesis, physicochemical properties, and pharmaceutical application of water-soluble CAs with emphasis on those that contain nitrogen-based substituents in their structure, particularly aminoCAs. In particular, it describes state-of-the-art in complexation of water-soluble CAs with pharmaceutically relevant ions and organic molecules up to amino acids, DNA, and proteins.