Johan Wouters

Copper‐mediated radiofluorination (CMRF) of arylstannanes has emerged as a powerful strategy for accessing fluorine‐18 labeled radiotracers. Yet, concerns regarding chemical impurity profiles remain insufficiently addressed. Here, we report the fully automated production of the clinically relevant synaptic vesicle glycoprotein 2A tracer [ ¹⁸ F]SynVesT‐1 on a GE FASTLab module, together with a systematic investigation of the associated impurity profile. Trimethyl‐ and tri(n‐butyl)stannane precursors, as well as nonradioactive analytical standards for all major putative impurities, were synthesized. Automated radiosynthesis afforded [ ¹⁸ F]SynVesT‐1 in 20%–25% nondecay‐corrected radiochemical yield, >99% radiochemical purity, and preserved enantiomeric excess. Comprehensive UPLC‐MS analysis revealed the formation of protodestannylated, hydroxylated, methoxylated, and dimeric side products. Notably, the methoxy‐substituted analog was identified as the major chemical impurity. Systematic variation of fluoride preprocessing conditions and precursor structure demonstrated that both residual methanol and trimethylstannyl leaving groups contribute to methoxylation. Replacement of methanol by n ‐butanol during fluoride processing, combined with a tri( n ‐butyl)stannane precursor, dramatically improved chemical purity. Beyond [ ¹⁸ F]SynVesT‐1, these findings underscore the critical importance of thoroughly investigating the influence of all reaction parameters on the impurity profile in CMRF‐based radiopharmaceutical production.

Bacterioferritin from Brucella melitensis ( Bm Bfr) is a protein that plays essential roles in oxidative stress management and metal homeostasis in the bacterium. It presents itself as a 24-mer forming a spherical nanocage, allowing further applications such as drug delivery and nanoparticle synthesis. Studying the oligomerization process is thus important for a deeper understanding of the protein. In this work, two forms of the protein were studied: with the heme cofactor (holo Bm Bfr) and without the cofactor (apo Bm Bfr). Analyses were performed using size-exclusion chromatography, thermal shift analysis and structure determination of apo Bm Bfr by X-ray crystallography. This revealed that oligomerization is not possible with the apo form, since the heme is essential for dimer formation, which is the first oligomerization step. Further oligomerization can occur at a pH above 5.0, where interface interactions are favorable, to form a highly stable 24-mer state. Those findings allowed us to suggest an oligomerization mechanism for Bm Bfr.

Our previous study demonstrated that bis-(1,2,3,4-tetrahydroisoquinoline) (bis-THIQ) compounds such as tetrandrine and AG525E1 exhibit significant affinity for SK channels. Interestingly, the stereoisomers of AG525 display markedly different affinities toward SK channels, with the (S,S) stereoisomer being the best ligand. Previous structure-based in silico investigations failed to provide a satisfactory explanation for these differences. Moreover, a series of simplified THIQ derivatives derived from AG525 showed little or no affinity for SK channels, raising questions about the structural features required for binding to SK channels. To better understand the structure–activity relationships within this chemical family with diverse three-dimensional features, a ligand-based approach is developed in this work. Using a dataset composed of AG525 stereoisomers and natural bis-THIQ derivatives, a pharmacophore model was generated to identify key molecular features required for binding to SK channels and to rationalize the observed differences in activity. Twenty pharmacophore hypotheses were generated, with the best-ranked models highlighting five major interaction features: an aromatic ring, one acceptor group associated with this ring, one lipophilic feature, one positive charge, and a second acceptor site located farther from the THIQ moiety. This model differs from previously proposed pharmacophores emphasizing the presence of two positive charges. Two alkaloids recently identified as SK channel ligands, (1) and (2), were found to match all five pharmacophoric features despite not being included in the training set. The absence of SK affinity of a series of simplified THIQ analogues derived from AG525 could be explained using the model. To go further, the predictive value of the model was evaluated by evaluating in silico the propensity of a series of cyclic and non-cyclic bis-THIQ alkaloids to bind to SK channels. High fitting scores were obtained for these compounds, and subsequent in vitro experiments, using radioligand competitive binding assays, were performed and confirmed that they belong as ligands.

The active form of lactate dehydrogenase B (LDHB) is a homotetramer, and disruption of this quaternary structure represents a promising strategy for therapeutic intervention, particularly in cancer metabolism. However, accurate characterization of LDHB oligomerization is challenging due to the weak, reversible, and environmentally sensitive nature of subunit interactions. Here, we establish a robust size exclusion chromatography coupled with multi angle light scattering (SEC–MALS) workflow for analysis of LDHB oligomerization under physiologically relevant conditions.Wild type LDHB and three single point interface mutants (I9A, L71A, and F72A), covering oligomeric states from tetramers to monomers, were analyzed. SEC–MALS enabled clear separation and absolute molar mass determination of coexisting oligomeric species at micromolar protein concentrations, capturing concentration dependent equilibria inaccessible to techniques requiring nanomolar levels. The wild type enzyme remained exclusively tetrameric and remarkably stable, whereas the mutants displayed heterogeneous, dynamic oligomeric distributions.Systematic evaluation of experimental parameters demonstrated that LDHB oligomerization is highly sensitive to buffer composition. While the wild type protein was largely unaffected, variations in salt nature, ionic strength, pH, organic solvent, and arginine selectively destabilized mutant assemblies. Sodium chloride emerged as the most appropriate salt for preserving native oligomeric states while minimizing ion specific perturbations.Together, these results establish SEC–MALS as a powerful and essential method for characterizing dynamic protein oligomerization equilibria under biologically relevant conditions and provide critical insights into the structural determinants governing LDHB assembly and stability.

The prolonged heating of an N -heterocyclic carbene-stabilised borenium cation resulted in the abstraction of a –C ₆ F ₅ group from its B(C ₆ F ₅ ) ₄ ⁻ counter-anion.

A novel imine‐based ligand ( E )‐2‐(4‐(((2‐hydroxynaphthalen‐1‐yl)methylene)amino)phenyl)acetonitrile was synthesized via mechanochemistry from 4‐aminobenzylcyanide and 2‐hydroxy‐1‐naphthaldehyde, achieving >99% yield. The ligand demonstrated exceptional Cu ²⁺ ion sensing through colorimetric detection, with a rapid visual response and a low detection limit (4.7 × 10 ^{− 8} M). Structural studies revealed 2:1 (L‐Cu ^{2 +} ) complex formation, confirmed by Job's plot and ¹ H NMR titration analyses. To enhance aqueous performance, host–guest complexation with β‐cyclodextrin was investigated. UV‐Vis and NMR spectroscopy confirmed 1:1 inclusion complex formation ( K _a = 6.2 × 10 ⁵ M ^{− 1} ) through aminobenzyl cyanide insertion into the β‐CD cavity. The L‐β‐CD system significantly improved selectivity by reducing Al ^{3 +} and Hg ^{2 +} interference while maintaining comparable sensitivity (LOD = 4.8 × 10 ^{− 8} M). Real‐world validation showed superior recovery rates (94.2%–110.0%) compared to free ligand (87.2%–114.7%). Biomolecular interaction studies revealed moderate DNA intercalation ( K _a = 1.2×10 ⁴ M ^{− 1} ) attenuated by β‐CD inclusion ( K _a = 3.5 × 10 ³ M ^{− 1} ), while bovine serum albumin (BSA) binding was slightly enhanced ( K _a increased from 2.2 × 10 ⁶ to 4.0 × 10 ⁶ M ^{− 1} ). This represents the UV‐Vis investigation of β‐cyclodextrin influence on Cu ^{2 +} sensing, providing insights for an advanced supramolecular analytical platform.

Copper-mediated radiofluorination (CMRF) of arylstannanes has emerged as a powerful strategy for accessing fluorine-18 labelled radiotracers. Yet concerns regarding chemical impurity profiles remain insufficiently addressed. Here, we report the fully automated production of the clinically relevant synaptic vesicle glycoprotein 2A tracer [¹⁸F]SynVesT-1 on a GE FASTLab module, together with a systematic investigation of the associated impurity profile. Trimethyl- and tri(n-butyl)stannane precursors, as well as non-radioactive analytical standards for all major putative impurities were synthesized. Automated radiosynthesis afforded [¹⁸F]SynVesT-1 in 20-25% non-decay-corrected radiochemical yield, >99% radiochemical purity, and with preserved enantiomeric excess. Comprehensive UPLC-MS analysis revealed the formation of protodesstannylated, hydroxylated, methoxylated, and dimeric side products. Notably, the methoxy-substituted analogue was identified as the major chemical impurity. Systematic variation of fluoride preprocessing conditions and precursor structure demonstrated that both residual methanol and trimethylstannyl leaving groups contribute to methoxylation. Replacement of methanol by n -butanol during fluoride processing, combined with a tri( n -butyl)stannane precursor, dramatically improved chemical purity. Beyond [¹⁸F]SynVesT-1, these findings underscore the critical importance of thoroughly investigating the influence of all reaction parameters on the impurity profile in CMRF-based radiopharmaceutical production.

The alternate O ‐methylation of p ‐ t Bu‐calix[6]arene is a well‐known method leading to the C _3v ‐symmetrical 1,3,5‐tris‐ O ‐Me‐ p ‐ t Bu‐calix[6]arene, a key compound for the development of platforms for supramolecular studies and in particular for the access to receptors in host–guest chemistry. The alternate O ‐methylation of the parent de‐ tert ‐butylated calix[6]arene remained elusive, precluding the development of similar C _3v ‐symmetrical 1,3,5‐tris‐ O ‐methylated receptors with an open cavity devoid of bulky t Bu groups at the large rim. In this work, we developed an efficient method for the synthesis of 1,3,5‐tris‐ O ‐Me‐calix[6]arene. This platform was further selectively derivatized on the small and large rims, producing notably a tris‐imidazole calix[6]arene‐based ligand forming a biomimetic funnel complex upon coordinating Zn ²⁺ _, with an open cavity suitable for the inclusion of a bulky dopamine derivative.