Resonance Raman and Visible Micro-Spectroscopy for the In-Vivo and In-Vitro Characterization of Anthocyanin-Based Pigments in Blue and Violet Flowers: A Comparison with HPLC-ESI- MS Analysis of the Extracts.

Microanalysis techniques based on resonance Raman and reflection visible spectroscopy have been applied to the characterization of pigments responsible for the blue or violet coloration in flowers; in particular of Lobelia erinus, Campanula portenschlagiana, Cineraria, Viola tricolor, Anemone coronaria, Agapanthus, Platycodon, Salvia farinacea, Plumbago capensis, Ceratostigma plumbaginoides, Commelina communis and Salvia patens. The spectroscopic methods were applied both in vivo on the flower petals and in vitro on extracts obtained through a procedure based on SPE (solid-phase extraction) optimized for minimal quantities of vegetable raw material. Different patterns obtained for the Raman spectra have been correlated, also on the basis of density functional theory (DFT) calculations, with different schemes of substitution of the benzopyrilium nucleus of the anthocyanins and with various possible forms of copigmentation responsible for the stabilization of the blue color. The results obtained were verified by comparison with the analysis of the extracts by HPLC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry).

Maytansinol Functionalization: Towards Useful Probes for Studying Microtubule Dynamics.

Invited for the cover of this issue are the groups of Professors Passarella and Pieraccini at the University of Milan, in collaboration with some of the members of TubInTrain consortium. The image depicts work with the elements of nature, in particular the destabilising effect of maytansinol (the constellation) on microtubules (the trees). Read the full text of the article at 10.1002/chem.202203431.

Maytansinol Functionalization: Towards Useful Probes for Studying Microtubule Dynamics.

Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.

Molecular Similarity Perception Based on Machine-Learning Models.

Molecular similarity is an impressively broad topic with many implications in several areas of chemistry. Its roots lie in the paradigm that 'similar molecules have similar properties'. For this reason, methods for determining molecular similarity find wide application in pharmaceutical companies, e.g., in the context of structure-activity relationships. The similarity evaluation is also used in the field of chemical legislation, specifically in the procedure to judge if a new molecule can obtain the status of orphan drug with the consequent financial benefits. For this procedure, the European Medicines Agency uses experts' judgments. It is clear that the perception of the similarity depends on the observer, so the development of models to reproduce the human perception is useful. In this paper, we built models using both 2D fingerprints and 3D descriptors, i.e., molecular shape and pharmacophore descriptors. The proposed models were also evaluated by constructing a dataset of pairs of molecules which was submitted to a group of experts for the similarity judgment. The proposed machine-learning models can be useful to reduce or assist human efforts in future evaluations. For this reason, the new molecules dataset and an online tool for molecular similarity estimation have been made freely available.

Maytansinol Derivatives: Side Reactions as a Chance for New Tubulin Binders.

Maytansinol is a valuable precursor for the preparation of maytansine derivatives (known as maytansinoids). Inspired by the intriguing structure of the macrocycle and the success in targeted cancer therapy of the derivatives, we explored the maytansinol acylation reaction. As a result, we were able to obtain a series of derivatives with novel modifications of the maytansine scaffold. We characterized these molecules by docking studies, by a comprehensive biochemical evaluation, and by determination of their crystal structures in complex with tubulin. The results shed further light on the intriguing chemical behavior of maytansinoids and confirm the relevance of this peculiar scaffold in the scenario of tubulin binders.

Exploring Orthogonality between Halogen and Hydrogen Bonding Involving Benzene.

The concept of orthogonality between halogen and hydrogen bonding, brought out by Ho and coworkers some years ago, has become a widely accepted idea within the chemists' community. While the original work was based on a common carbonyl oxygen as acceptor for both interactions, we explore here, by means of M06-2X, M11, ωB97X, and ωB97XD/aug-cc-PVTZ DFT calculations, the interdependence of halogen and hydrogen bonding with a shared π-electron system of benzene. The donor groups (specifically NCBr and H2O) were placed on either or the same side of the ring, according to a double T-shaped or a perpendicular geometry, respectively. The results demonstrate that the two interactions with benzene are not strictly independent on each other, therefore outlining that the orthogonality between halogen and hydrogen bonding, intended as energetical independence between the two interactions, should be carefully evaluated according to the specific acceptor group.

Simulating Multiple Substrate-Binding Events by γ-Glutamyltransferase Using Accelerated Molecular Dynamics.

γ-Glutamyltransferase (GGT) is an enzyme that uses γ-glutamyl compounds as substrates and catalyzes their transfer to a water molecule or an acceptor substrate with varied physiological function in bacteria, plants, and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, the structural dynamics of the substrate binding to the catalytic site of GGT are unknown. Here, we modeled Escherichia coli GGT's glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggest its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

Modelling of short synthetic antifreeze peptides: Insights into ice-pinning mechanism.

Organisms living in icy environments produce antifreeze proteins to control ice growth and recrystallization. It has been proposed that these molecules pin the surface of ice crystals, thus inducing the formation of a curved surface that arrests crystal growth. Such proteins are very appealing for many potential applications in food industry, material science and cryoconservation of organs and tissues. Unfortunately, their structural complexity has seriously hampered their practical use, while efficient and accessible synthetic analogues are highly desirable. In this paper, we used molecular dynamics based techniques to model the interaction of three short antifreeze synthetic peptides with an ice surface. The employed protocols succeeded in reproducing the ice pinning action of antifreeze peptides and the consequent ice growth arrest, as well as in distinguishing between antifreeze and control peptides, for which no such effect was observed. Principal components analysis of peptides trajectories in different simulation settings permitted to highlight the main structural features associated to antifreeze activity. Modeling results are highly correlated with experimentally measured properties, and insights on ice-peptide interactions and on conformational patterns favoring antifreeze activity will prompt the design of new and improved antifreeze peptides.

The Origin of the σ-Hole in Halogen Atoms: a Valence Bond Perspective.

A detailed Valence Bond-Spin Coupled analysis of a series of halogenated molecules is here reported, allowing to get a rigorous ab initio demonstration of the qualitative models previously proposed to explain the origin of halogen bonding. The concepts of σ-hole and negative belt observed around the halogen atoms in the electrostatic potential maps are here interpreted by analysis of the relevant Spin Coupled orbitals.

On-resin multicomponent 1,3-dipolar cycloaddition of cyclopentanone-proline enamines and sulfonylazides as an efficient tool for the synthesis of amidino depsipeptide mimics.

Depsipeptides are biologically active peptide derivatives that possess a high therapeutic interest. The development of depsipeptide mimics characterized by a chemical diversity could lead to compounds with enhanced features and activity. In this work, an on-resin multicomponent procedure for the synthesis of amidino depsipeptide mimics is described. This approach exploits a metal-free 1,3-dipolar cycloaddition of cyclopentanone-proline enamines and sulfonylazides. In this reaction, the obtained primary cycloadduct undergoes a ring opening and molecular rearrangement giving access to a linear sulfonyl amidine functionalized with both a peptide chain and a diazoalkane. The so-obtained diazo function "one pot" reacts with the carboxylic group of N-Fmoc-protected amino acids leading to amidino depsipeptide mimics possessing a C4 aliphatic chain. An important advantage of this procedure is the possibility to easily obtain amidino-functionalized derivatives that are proteolytically stable peptide bond bioisosteres. Moreover, the conformational freedom given by the alkyl chain could promote the obtainment of cyclic depsipeptide with a stabilized secondary structure as demonstrated with both in silico calculations and experimental conformational studies. Finally, labeled depsipeptide mimics can be also synthesized using a fluorescent sulfonylazide in the multicomponent reaction.

X-ray constrained spin-coupled technique: theoretical details and further assessment of the method.

One of the well-established methods of modern quantum crystallography is undoubtedly the X-ray constrained wavefunction (XCW) approach, a technique that enables the determination of wavefunctions which not only minimize the energy of the system under examination, but also reproduce experimental X-ray diffraction data within the limit of the experimental errors. Initially proposed in the framework of the Hartree-Fock method, the strategy has been gradually extended to other techniques of quantum chemistry, but always remaining limited to a single-determinant ansatz for the wavefunction to extract. This limitation has been recently overcome through the development of the novel X-ray constrained spin-coupled (XCSC) approach [Genoni et al. (2018). Chem. Eur. J. 24, 15507-15511] which merges the XCW philosophy with the traditional spin-coupled strategy of valence bond theory. The main advantage of this new technique is the possibility of extracting traditional chemical descriptors (e.g. resonance structure weights) compatible with the experimental diffraction measurements, without the need to introduce information a priori or perform analyses a posteriori. This paper provides a detailed theoretical derivation of the fundamental equations at the basis of the XCSC method and also introduces a further advancement of its original version, mainly consisting in the use of molecular orbitals resulting from XCW calculations at the Hartree-Fock level to describe the inactive electrons in the XCSC computations. Furthermore, extensive test calculations, which have been performed by exploiting high-resolution X-ray diffraction data for salicylic acid and by adopting different basis sets, are presented and discussed. The computational tests have shown that the new technique does not suffer from particular convergence problems. Moreover, all the XCSC calculations provided resonance structure weights, spin-coupled orbitals and global electron densities slightly different from those resulting from the corresponding unconstrained computations. These discrepancies can be ascribed to the capability of the novel strategy to capture the information intrinsically contained in the experimental data used as external constraints.

The selective disruption of presynaptic JNK2/STX1a interaction reduces NMDA receptor-dependent glutamate release.

The neuronal loss caused by excessive glutamate release, or 'excitotoxicity', leads to several pathological conditions, including cerebral ischemia, epilepsy, and neurodegenerative diseases. Over-stimulation of presynaptic N-methyl-D-aspartate (NMDA) receptors is known to trigger and support glutamate spillover, while postsynaptic NMDA receptors are responsible for the subsequent apoptotic cascade. Almost all molecules developed so far are unable to selectively block presynaptic or postsynaptic NMDA receptors, therefore a deeper knowledge about intracellular NMDA pathways is required to design more specific inhibitors. Our previous work showed that presynaptic c-Jun N-terminal kinase 2 (JNK2) specifically regulates NMDA-evoked glutamate release and here we demonstrate that an interaction between Syntaxin-1a and JNK2 is fundamental to this mechanism. Based on this evidence, a new cell permeable peptide (CPP), "JGRi1", has been developed to disrupt the JNK2/STX1a interaction to indirectly, but specifically, inhibit presynaptic NMDA receptor signaling. JGRi1 reduces the NMDA-evoked release of glutamate both in in-vitro and ex-vivo experiments while also being able to widely diffuse throughout brain tissue via intraperitoneal administration. In conclusion, the JNK2/STX1 interaction is involved in presynaptic NMDA-evoked glutamate release and the novel CPP, JGRi1, acts as a pharmacological tool that promotes neuroprotection.

Synthesis of Thicolchicine-Based Conjugates: Investigation towards Bivalent Tubulin/Microtubules Binders.

Four different hybrid compounds have been efficiently synthesized by conjugation of deacetylthiocolchicine with pironetin-inspired derivatives. The modest bioactivity and the apparent absence of interaction with α-tubulin is explained by a posteriori in silico investigation, which suggests a relevant distance between the thiocolchicine binding site and the proper pocket on the α-tubulin. The modest activity on resistant cells suggested that the lipophilic nature of the linker used renders the resulting compounds better substrates for p-Gp efflux pumps. The study better clarifies the design of bivalent compounds that target hetero tubulin/microtubules.

Molecular Dynamics Simulations of p97 Including Covalent, Allosteric and ATP-competitive Inhibitors.

Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of action.

On the molecular optical nonlinearity of halogen-bond-forming azobenzenes.

We study hyper-Rayleigh scattering and computed molecular hyperpolarizability in a series of azobenzene chromophores in chloroform and dimethylformamide as solvents. The chromophores form halogen or hydrogen bonds of varying strength with dimethylformamide molecules, differently from what is expected for chloroform. We show that hyperpolarizability is unaffected or sligthly lower with the azobenzene forming the strongest halogen bond. Solid supramolecular polymers with the same chromophores have previously demonstrated clearly higher second-order nonlinear responses when a halogen-bond-accepting polymer is used, the larger increase being associated with the stronger halogen bond. The present study proves that the higher optical nonlinearity in polymers lies in the better ordering of the chromophores instead of changes in molecular hyperpolarizability, highlighting the unique properties of halogen bonding in supramolecular chemistry.

X-ray Constrained Spin-Coupled Wavefunction: a New Tool to Extract Chemical Information from X-ray Diffraction Data.

The X-ray constrained wavefunction (XCW) approach is a reliable and widely used method of quantum crystallography that allows the determination of wavefunctions compatible with X-ray diffraction data. So far, all the existing XCW techniques have been developed in the framework of molecular orbital theory and, consequently, provide only pictures of the "experimental" electronic structures that are far from the traditional chemical perception. Here a new strategy is proposed that, by combining the XCW philosophy with the spin-coupled method of valence bond theory, enables direct extraction of traditional chemical information (e.g., weights of resonance structures) from X-ray diffraction measurements. Preliminary results have shown that the new technique is really able to efficiently capture the effects of the crystal environment on the electronic structure, and can be considered as a new useful tool to perform chemically sound analyses of the X-ray diffraction data.

Imidazo[2,1-b]benzothiazol Derivatives as Potential Allosteric Inhibitors of the Glucocorticoid Receptor.

Glucocorticoid receptor (GCR) transactivation reporter gene assays were used as an initial high-throughput screening on a diversified library of 1200 compounds for their evaluation as GCR antagonists. A class of imidazo[2,1-b]benzothiazole and imidazo[2,1-b]benzoimidazole derivatives were identified for their ability to modulate GCR transactivation and anti-inflammatory transrepression effects utilizing GCR and NF-κB specific reporter gene assays. Modeling studies on the crystallographic structure of the GCR ligand binding domain provided three new analogues bearing the tetrahydroimidazo[2,1-b]benzothiazole scaffold able to antagonize the GCR in the presence of dexamethasone (DEX) and also defined their putative binding into the GCR structure. Both mRNA level measures of GCR itself and its target gene GILZ, on cells treated with the new analogues, showed a GCR transactivation inhibition, thus suggesting a potential allosteric inhibition of the GCR.

Assessment of DFT Functionals for QTAIM Topological Analysis of Halogen Bonds with Benzene.

Halogen bonding, a noncovalent interaction between a halogen atom and a nucleophilic site, is receiving a growing attention in the chemical community stimulating a large number of theoretical investigations. The density functional theory (DFT) approach revealed to be one of the most suitable methods owing to its accuracy and low computational cost. We report here a detailed analysis of the performance of an extensive set of DFT functionals in reproducing accurate binding energies and topological properties for the halogen-bonding interaction of either NCX or PhX molecules (X = F, Cl, Br, I) with the aromatic system of benzene in the T-shaped configuration. It was found that the better performance for both sets of properties is provided by a small subset of functionals able to take into account, implicitly or explicitly (by inclusion of an additive pairwise potential), the dispersion contribution, that is, ωB97X, M06-2X, M11, mPW2PLYP-D, and B2PLYP-D3.

4-(1,2-diarylbut-1-en-1-yl)isobutyranilide derivatives as inhibitors of topoisomerase II.

The synthesis and biological evaluation of a new library of 4-(1,2-diarylbut-1-en-1-yl)isobutyranilides is described. The new compounds were found to be cytotoxic in the micromolar range in two human tumor cell lines, MCF-7 (mammary gland adenocarcinoma) and HeLa (cervix adenocarcinoma) and two human ovarian cancer cell lines (A2780 and OVCAR5). Detailed studies on the most active compound 6g show that it was able to induce apoptosis and suggest topoisomerase II as a possible intracellular target. The relevance of the interaction of the most active compound with topoisomerase II is demonstrated and supported by docking studies.

Boehmeriasin A as new lead compound for the inhibition of topoisomerases and SIRT2.

Two synthetic approaches to boehmeriasin A are described. A gram scale racemic preparation is accompanied by an efficient preparation of both the pure enantiomers using the conformationally stable 2-piperidin-2-yl acetaldehyde as starting material. The anti-proliferative activity in three cancer cell lines (CEM, HeLa and L1210) and two endothelial cell lines (HMEC-1, BAEC) indicates promising activity at the nanomolar range. Topoisomerases and SIRT2 are identified as biological targets and the experimental data has been supported by docking studies.